Human ion transport-like protein

ABSTRACT

The invention provides a human ion transport-like protein (HITLP) and polynucleotides which identify and encode HITLP. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for diagnosing, treating, or preventing disorders associated with expression of HITLP.

FIELD OF THE INVENTION

This invention relates to nucleic acid and amino acid sequences of ahuman ion transport-like protein and to the use of these sequences inthe diagnosis, treatment, and prevention of osmoregulatory andinflammatory disorders.

BACKGROUND OF THE INVENTION

Osmoregulation occurs in all organisms, though the mechanisms differaccording to the organism's environment. Fresh water inhabitants need toretain salts, whereas ocean inhabitants need to retain water.Terrestrial inhabitants need to conserve both water and salts. Organismsmust balance these needs with a requirement to eliminate metabolicwaste, such as nitrogenous waste, and generate secreted body fluids,such as saliva for digestion and sweat for thermoregulation.

In mammals, sweat glands, salivary glands, and the kidney all produce aprimary secretion that is essentially isosmotic with blood andextracellular fluids. Modification of this primary secretion then occursas much of the sodium chloride and water are reabsorbed as they passthrough the excretory ducts of the glands and kidney, whereas potassiumand bicarbonate ions are secreted. This modification of the primarysecretion is important in the sweat glands to conserve sodium chloridein hot environments, and in the salivary glands to conserve sodiumchloride when excessive quantities of saliva are lost. This modificationis critical in the kidney to maintain proper sodium and water balance inthe extracellular fluids, a balance which also regulates arterialpressure. Loss of this modification activity by the duct cells causes alarge loss of sodium and water, resulting in severe dehydration and lowblood volume, and ultimately to circulatory collapse.

Sodium absorption by the intestines, especially in the colon, isnecessary to prevent loss of sodium in the stools. The loss of sodiumabsorption produces a failure to absorb anions and water as well. Theunabsorbed sodium chloride and water then lead to diarrhea, with furtherloss of sodium chloride from the body. Other body fluids may be underregulation similar to that seen in the systems described above. Forexample, cerebrospinal fluid is produced by active sodium ion transportfrom the capillaries across the epithelium of the choroid plexus, whichin turn attracts chloride ions and water. A counter flow of potassiumand bicarbonate ions move out of the cerebrospinal fluid into thecapillaries. A dysfunction in osmoregulation is associated with severaldisease states, including hyponatremia, renal failure, andhypernatremia. (Strange, K. (1992) J Am. Soc. Nephrol. 3:12-27.)

In insects, a system of osmoregulation similar to that in mammalsexists. The malpighian tubules are blind-ended sacs extending from thejuncture of the midgut and hindgut into the fluid-filled body cavity,the hemocoel. Active transport of potassium ions from the body fluid,the hemolymph, with passive diffusion of other ions and solutes, intothe lumen of the malpighian tubule produces a urine essentiallyisosmotic relative to hemolymph. The urine then passes into the gut,where selective, controlled reabsorption of essential solutes and mostof the water occurs in the anterior (ileum) and posterior (rectum)hindgut.

The insect ileum is functionally analogous to the proximal tubules ofthe vertebrate kidney. Reabsorption of fluids occurs due to activetransport of chloride and sodium ions, with a passive flow of potassiumions, from the ilium to the lumen of the malpighian tubule. Also,hydrogen ions and NH₄ ⁺ secretion, associated with bicarbonateabsorption, contributes to pH regulation and nitrogen excretion bymechanisms which are analogous to those in the vertebrate kidneyproximal tubules. (Ring, M. et al. (1998) Insect Biochem. Molec. Biol.28:51-58.) A neuropeptide secreted by the corpus cardiacum in the locustSchistocerca gregaria appears to regulate ileal ion transport. The iontransport peptide (ITP), at picomole levels, fully stimulates Cl, Na⁺,and K⁺ movement and fluid reabsorption in the locust ileum. ITP alsoinhibits ileum acid secretion almost completely. Cyclic adenosinemonophosphate (cAMP) also can stimulate ion movement and fluidreabsorption in the locust ileum, suggesting that ITP may work throughthis second messenger. (Meredith, J. et al. (1996) J. Exp. Biol.199:1053-1061.)

ITP is produced as a prepropeptide of 130 amino acids. Cleavage of thefirst 55 N-terminal amino acids and the last C-terminal amino acidresults in the native ITP molecule. ITP has considerable sequencesimilarity to a family of crustacean hormones including the crustaceanhyperglycemic hormone (CHH), molt inhibiting hormone (MIH), andvitellogenesis-inhibiting hormone (VIH). ITP shows 42% homology withCHH. ITP and CHH also share six conserved cysteine residues common toall of the crustacean hormone family members and a potential amidationsite at the C-terminal end of the molecule. Cleavage of the C-terminalend followed by amidation of the carboxyl group is a common maturationpathway for physiologically active peptides. (Meredith, supra; Soyez, D.(1997) Annals N Y Acad. Sci. 814:319-323.)

The discovery of a new human ion transport-like protein and thepolynucleotides encoding it satisfies a need in the art by providing newcompositions which are useful in the diagnosis, treatment, andprevention of osmoregulatory and inflammatory disorders.

SUMMARY OF THE INVENTION

The invention is based on the discovery of a new human iontransport-like protein (HITLP), the polynucleotides encoding HITLP, andthe use of these compositions for the diagnosis, treatment, orprevention of osmoregulatory and inflammatory disorders.

The invention features a substantially purified polypeptide comprisingthe amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.

The invention further provides a substantially purified variant havingat least 90% amino acid sequence identity to the amino acid sequence ofSEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention also provides anisolated and purified polynucleotide encoding the polypeptide comprisingthe sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The inventionalso includes an isolated and purified polynucleotide variant having atleast 70% polynucleotide sequence identity to the polynucleotideencoding the polypeptide comprising the amino acid sequence of SEQ IDNO:1 or a fragment of SEQ ID NO:1.

The invention further provides an isolated and purified polynucleotidewhich hybridizes under stringent conditions to the polynucleotideencoding the polypeptide comprising the amino acid sequence of SEQ IDNO:1 or a fragment of SEQ ID NO:1, as well as an isolated and purifiedpolynucleotide which is complementary to the polynucleotide encoding thepolypeptide comprising the amino acid sequence of SEQ ID NO:1 or afragment of SEQ ID NO:1.

The invention also provides an isolated and purified polynucleotidecomprising the polynucleotide sequence of SEQ ID NO:2 or a fragment ofSEQ ID NO:2, and an isolated and purified polynucleotide variant havingat least 70% polynucleotide sequence identity to the polynucleotidecomprising the polynucleotide sequence of SEQ ID NO:2 or a fragment ofSEQ ID NO:2. The invention also provides an isolated and purifiedpolynucleotide having a sequence complementary to the polynucleotidecomprising the polynucleotide sequence of SEQ ID NO:2 or a fragment ofSEQ ID NO:2.

The invention further provides an expression vector comprising at leasta fragment of the polynucleotide encoding the polypeptide comprising thesequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. In another aspect,the expression vector is contained within a host cell.

The invention also provides a method for producing a polypeptidecomprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQID NO:1, the method comprising the steps of: (a) culturing the host cellcomprising an expression vector containing at least a fragment of apolynucleotide encoding the polypeptide comprising the amino acidsequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 under conditionssuitable for the expression of the polypeptide; and (b) recovering thepolypeptide from the host cell culture.

The invention also provides a pharmaceutical composition comprising asubstantially purified polypeptide having the sequence of SEQ ID NO:1 ora fragment of SEQ ID NO:1 in conjunction with a suitable pharmaceuticalcarrier.

The invention further includes a purified antibody which binds to apolypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQID NO:1, as well as a purified agonist and a purified antagonist of thepolypeptide.

The invention also provides a method for treating or preventing anosmoregulatory disorder, the method comprising administering to asubject in need of such treatment an effective amount of apharmaceutical composition comprising a substantially purifiedpolypeptide having the amino acid sequence of SEQ ID NO:1 or a fragmentof SEQ ID NO:1.

The invention also provides a method for treating or preventing aninflammatory disorder, the method comprising administering to a subjectin need of such treatment an effective amount of a pharmaceuticalcomposition comprising a substantially purified polypeptide having theamino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.

The invention also provides a method for detecting a polynucleotideencoding a polypeptide comprising the amino acid sequence of SEQ ID NO:1or a fragment of SEQ ID NO:1 in a biological sample containing nucleicacids, the method comprising the steps of: (a) hybridizing thecomplement of the polynucleotide encoding the polypeptide comprising theamino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 to atleast one of the nucleic acids of the biological sample, thereby forminga hybridization complex; and (b) detecting the hybridization complex,wherein the presence of the hybridization complex correlates with thepresence of a polynucleotide encoding the polypeptide comprising theamino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 in thebiological sample. In one aspect, this method further comprisesamplifying the polynucleotide prior to hybridization.

BRIEF DESCRIPTION OF THE FIGURES AND TABLE

FIGS. 1A and 1B show the amino acid sequence (SEQ ID NO:1) and nucleicacid sequence (SEQ ID NO:2) of HITLP. The alignment was produced usingMacDNASIS PRO™ software (Hitachi Software Engineering Co. Ltd., SanBruno, Calif.).

FIG. 2 shows the amino acid sequence alignments among HITLP (IncyteClone 3171334; SEQ ID NO:1), locust ion transport protein (GI 1244522;SEQ ID NO:3), and locust ion transport-like protein (GI 1244524; SEQ IDNO:4), produced using the multisequence alignment program of LASERGENE™software (DNASTAR Inc, Madison Wis.).

Table 1 shows the programs, their descriptions, references, andthreshold parameters used to identify and characterize HITLP.

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleotide sequences, and methods aredescribed, it is understood that this invention is not limited to theparticular methodology, protocols, cell lines, vectors, and reagentsdescribed, as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms "a," "an," and "the" include plural reference unless thecontext clearly dictates otherwise. Thus, for example, a reference to "ahost cell" includes a plurality of such host cells, and a reference to"an antibody" is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are now described. All publications mentionedherein are cited for the purpose of describing and disclosing the celllines, vectors, and methodologies which are reported in the publicationsand which might be used in connection with the invention. Nothing hereinis to be construed as an admission that the invention is not entitled toantedate such disclosure by virtue of prior invention.

DEFINITIONS

"HITLP," as used herein, refers to the amino acid sequences, or variantthereof, of substantially purified HITLP obtained from any species,particularly a mammalian species, including bovine, ovine, porcine,murine, equine, and preferably the human species, from any source,whether natural, synthetic, semi-synthetic, or recombinant.

The term "agonist," as used herein, refers to a molecule which, whenbound to HITLP, increases or prolongs the duration of the effect ofHITLP. Agonists may include proteins, nucleic acids, carbohydrates, orany other molecules which bind to and modulate the effect of HITLP.

An "allelic variant," as this term is used herein, is an alternativeform of the gene encoding HITLP. Allelic variants may result from atleast one mutation in the nucleic acid sequence and may result inaltered mRNAs or in polypeptides whose structure or function may or maynot be altered. Any given natural or recombinant gene may have none,one, or many allelic forms. Common mutational changes which give rise toallelic variants are generally ascribed to natural deletions, additions,or substitutions of nucleotides. Each of these types of changes mayoccur alone, or in combination with the others, one or more times in agiven sequence.

"Altered" nucleic acid sequences encoding HITLP, as described herein,include those sequences with deletions, insertions, or substitutions ofdifferent nucleotides, resulting in a polynucleotide the same as HITLPor a polypeptide with at least one functional characteristic of HITLP.Included within this definition are polymorphisms which may or may notbe readily detectable using a particular oligonucleotide probe of thepolynucleotide encoding HITLP, and improper or unexpected hybridizationto allelic variants, with a locus other than the normal chromosomallocus for the polynucleotide sequence encoding HITLP. The encodedprotein may also be "altered," and may contain deletions, insertions, orsubstitutions of amino acid residues which produce a silent change andresult in a functionally equivalent HITLP. Deliberate amino acidsubstitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues, as long as the biological orimmunological activity of HITLP is retained. For example, negativelycharged amino acids may include aspartic acid and glutamic acid,positively charged amino acids may include lysine and arginine, andamino acids with uncharged polar head groups having similarhydrophilicity values may include leucine, isoleucine, and valine;glycine and alanine; asparagine and glutamine; serine and threonine; andphenylalanine and tyrosine.

The terms "amino acid" or "amino acid sequence," as used herein, referto an oligopeptide, peptide, polypeptide, or protein sequence, or afragment of any of these, and to naturally occurring or syntheticmolecules. In this context, "fragments," "immunogenic fragments," or"antigenic fragments" refer to fragments of HITLP which are preferablyat least 5 to about 15 amino acids in length, most preferably at least14 amino acids, and which retain some biological activity orimmunological activity of HITLP. Where "amino acid sequence" is recitedherein to refer to an amino acid sequence of a naturally occurringprotein molecule, "amino acid sequence" and like terms are not meant tolimit the amino acid sequence to the complete native amino acid sequenceassociated with the recited protein molecule.

"Amplification," as used herein, relates to the production of additionalcopies of a nucleic acid sequence. Amplification is generally carriedout using polymerase chain reaction (PCR) technologies well known in theart. (See, e.g., Dieffenbach, C. W. and G. S. Dveksler (1995) PCRPrimer, a Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.,pp. 1-5.)

The term "antagonist," as it is used herein, refers to a molecule which,when bound to HITLP, decreases the amount or the duration of the effectof the biological or immunological activity of HITLP. Antagonists mayinclude proteins, nucleic acids, carbohydrates, antibodies, or any othermolecules which decrease the effect of HITLP.

As used herein, the term "antibody" refers to intact molecules as wellas to fragments thereof, such as Fab, F(ab')₂, and Fv fragments, whichare capable of binding the epitopic determinant. Antibodies that bindHITLP polypeptides can be prepared using intact polypeptides or usingfragments containing small peptides of interest as the immunizingantigen. The polypeptide or oligopeptide used to immunize an animal(e.g., a mouse, a rat, or a rabbit) can be derived from the translationof RNA, or synthesized chemically, and can be conjugated to a carrierprotein if desired. Commonly used carriers that are chemically coupledto peptides include bovine serum albumin, thyroglobulin, and keyholelimpet hemocyanin (KLH). The coupled peptide is then used to immunizethe animal.

The term "antigenic determinant," as used herein, refers to thatfragment of a molecule (i.e., an epitope) that makes contact with aparticular antibody. When a protein or a fragment of a protein is usedto immunize a host animal, numerous regions of the protein may inducethe production of antibodies which bind specifically to antigenicdeterminants (given regions or three-dimensional structures on theprotein). An antigenic determinant may compete with the intact antigen(i.e., the immunogen used to elicit the immune response) for-binding toan antibody.

The term "antisense," as used herein, refers to any compositioncontaining a nucleic acid sequence which is complementary to the "sense"strand of a specific nucleic acid sequence. Antisense molecules may beproduced by any method including synthesis or transcription. Onceintroduced into a cell, the complementary nucleotides combine withnatural sequences produced by the cell to form duplexes and to blockeither transcription or translation. The designation "negative" canrefer to the antisense strand, and the designation "positive" can referto the sense strand.

As used herein, the term "biologically active," refers to a proteinhaving structural, regulatory, or biochemical functions of a naturallyoccurring molecule. Likewise, "immunologically active" refers to thecapability of the natural, recombinant, or synthetic HITLP, or of anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

The terms "complementary" or "complementarity," as used herein, refer tothe natural binding of polynucleotides by base pairing. For example, thesequence "5'A-G-T 3'" binds to the complementary sequence "3'T-C-A 5'."Complementarity between two single-stranded molecules may be "partial,"such that only some of the nucleic acids bind, or it may be "complete,"such that total complementarity exists between the single strandedmolecules. The degree of complementarity between nucleic acid strandshas significant effects on the efficiency and strength of thehybridization between the nucleic acid strands. This is of particularimportance in amplification reactions, which depend upon binding betweennucleic acids strands, and in the design and use of peptide nucleic acid(PNA) molecules.

A "composition comprising a given polynucleotide sequence" or a"composition comprising a given amino acid sequence," as these terms areused herein, refer broadly to any composition containing the givenpolynucleotide or amino acid sequence. The composition may comprise adry formulation or an aqueous solution. Compositions comprisingpolynucleotide sequences encoding HITLP or fragments of HITLP may beemployed as hybridization probes. The probes may be stored infreeze-dried form and may be associated with a stabilizing agent such asa carbohydrate. In hybridizations, the probe may be deployed in anaqueous solution containing salts, e.g., NaCl, detergents, e.g.,sodiumdodecyl sulfate (SDS), and other components, e.g., Denhardt's solution,dry milk, salmon sperm DNA, etc.

"Consensus sequence," as used herein, refers to a nucleic acid sequencewhich has been resequenced to resolve uncalled bases, extended usingXL-PCR™ (The Perkin-Elmer Corp., Norwalk, Conn.) in the 5' and/or the 3'direction, and resequenced, or which has been assembled from theoverlapping sequences of more than one Incyte Clone using a computerprogram for fragment assembly, such as the GELVIEW™ Fragment Assemblysystem (GCG, Madison, Wis.). Some sequences have been both extended andassembled to produce the consensus sequence.

As used herein, the term "correlates with expression of apolynucleotide" indicates that the detection of the presence of nucleicacids, the same or related to a nucleic acid sequence encoding HITLP, byNorthern analysis is indicative of the presence of nucleic acidsencoding HITLP in a sample, and thereby correlates with expression ofthe transcript from the polynucleotide encoding HITLP.

A "deletion," as the term is used herein, refers to a change in theamino acid or nucleotide sequence that results in the absence of one ormore amino acid residues or nucleotides.

The term "derivative," as used herein, refers to the chemicalmodification of a polypeptide sequence, or a polynucleotide sequence.Chemical modifications of a polynucleotide sequence can include, forexample, replacement of hydrogen by an alkyl, acyl, or amino group. Aderivative polynucleotide encodes a polypeptide which retains at leastone biological or immunological function of the natural molecule. Aderivative polypeptide is one modified by glycosylation, pegylation, orany similar process that retains at least one biological orimmunological function of the polypeptide from which it was derived.

The term "similarity," as used herein, refers to a degree ofcomplementarity. There may be partial similarity or complete similarity.The word "identity" may substitute for the word "similarity." Apartially complementary sequence that at least partially inhibits anidentical sequence from hybridizing to a target nucleic acid is referredto as "substantially similar." The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or Northern blot, solutionhybridization, and the like) under conditions of reduced stringency. Asubstantially similar sequence or hybridization probe will compete forand inhibit the binding of a completely similar (identical) sequence tothe target sequence under conditions of reduced stringency. This is notto say that conditions of reduced stringency are such that non-specificbinding is permitted, as reduced stringency conditions require that thebinding of two sequences to one another be a specific (i.e., aselective) interaction. The absence of non-specific binding may betested by the use of a second target sequence which lacks even a partialdegree of complementarity (e.g., less than about 30% similarity oridentity). In the absence of non-specific binding, the substantiallysimilar sequence or probe will not hybridize to the secondnon-complementary target sequence.

The phrases "percent identity" or "% identity" refer to the percentageof sequence similarity found in a comparison of two or more amino acidor nucleic acid sequences. Percent identity can be determinedelectronically, e.g., by using the MegAlign™ program (DNASTAR, Inc.,Madison Wis.). The MegAlign™ program can create alignments between twoor more sequences according to different methods, e.g., the clustalmethod. (See, e.g., Higgins, D. G. and P. M. Sharp (1988) Gene73:237-244.) The clustal algorithm groups sequences into clusters byexamining the distances between all pairs. The clusters are alignedpairwise and then in groups. The percentage similarity between two aminoacid sequences, e.g., sequence A and sequence B, is calculated bydividing the length of sequence A, minus the number of gap residues insequence A, minus the number of gap residues in sequence B, into the sumof the residue matches between sequence A and sequence B, times onehundred. Gaps of low or of no similarity between the two amino acidsequences are not included in determining percentage similarity. Percentidentity between nucleic acid sequences can also be counted orcalculated by other methods known in the art, e.g., the Jotun Heinmethod. (See, e.g., Hein, J. (1990) Methods Enzymol. 183:626-645.)Identity between sequences can also be determined by other methods knownin the art, e.g., by varying hybridization conditions.

"Human artificial chromosomes" (HACs), as described herein, are linearmicrochromosomes which may contain DNA sequences of about 6 kb to 10 Mbin size, and which contain all of the elements required for stablemitotic chromosome segregation and maintenance. (See, e.g., Harrington,J. J. et al. (1997) Nat Genet. 15:345-355.)

The term "humanized antibody," as used herein, refers to antibodymolecules in which the amino acid sequence in the non-antigen bindingregions has been altered so that the antibody more closely resembles ahuman antibody, and still retains its original binding ability.

"Hybridization," as the term is used herein, refers to any process bywhich a strand of nucleic acid binds with a complementary strand throughbase pairing.

As used herein, the term "hybridization complex" refers to a complexformed between two nucleic acid sequences by virtue of the formation ofhydrogen bonds between complementary bases. A hybridization complex maybe formed in solution (e.g., C₀ t or R₀ t analysis) or formed betweenone nucleic acid sequence present in solution and another nucleic acidsequence immobilized on a solid support (e.g., paper, membranes,filters, chips, pins or glass slides, or any other appropriate substrateto which cells or their nucleic acids have been fixed).

The words "insertion" or "addition," as used herein, refer to changes inan amino acid or nucleotide sequence resulting in the addition of one ormore amino acid residues or nucleotides, respectively, to the sequencefound in the naturally occurring molecule.

"Immune response" can refer to conditions associated with inflammation,trauma, immune disorders, or infectious or genetic disease, etc. Theseconditions can be characterized by expression of various factors, e.g.,cytokines, chemokines, and other signaling molecules, which may affectcellular and systemic defense systems.

The term "microarray," as used herein, refers to an arrangement ofdistinct polynucleotides arrayed on a substrate, e.g., paper, nylon orany other type of membrane, filter, chip, glass slide, or any othersuitable solid support.

The terms "element" or "array element" as used herein in a microarraycontext, refer to hybridizable polynucleotides arranged on the surfaceof a substrate.

The term "modulate," as it appears herein, refers to a change in theactivity of HITLP. For example, modulation may cause an increase or adecrease in protein activity, binding characteristics, or any otherbiological, functional, or immunological properties of HITLP.

The phrases "nucleic acid" or "nucleic acid sequence," as used herein,refer to a nucleotide, oligonucleotide, polynucleotide, or any fragmentthereof. These phrases also refer to DNA or RNA of genomic or syntheticorigin which may be single-stranded or double-stranded and may representthe sense or the antisense strand, to peptide nucleic acid (PNA), or toany DNA-like or RNA-like material. In this context, "fragments" refersto those nucleic acid sequences which, when translated, would producepolypeptides retaining some functional characteristic, e.g.,antigenicity, or structural domain characteristic, e.g., ATP-bindingsite, of the full-length polypeptide.

The terms "operably associated" or "operably linked," as used herein,refer to functionally related nucleic acid sequences. A promoter isoperably associated or operably linked with a coding sequence if thepromoter controls the translation of the encoded polypeptide. Whileoperably associated or operably linked nucleic acid sequences can becontiguous and in the same reading frame, certain genetic elements,e.g., repressor genes, are not contiguously linked to the sequenceencoding the polypeptide but still bind to operator sequences thatcontrol expression of the polypeptide.

The term "oligonucleotide," as used herein, refers to a nucleic acidsequence of at least about 6 nucleotides to 60 nucleotides, preferablyabout 15 to 30 nucleotides, and most preferably about 20 to 25nucleotides, which can be used in PCR amplification or in ahybridization assay or microarray. As used herein, the term"oligonucleotide" is substantially equivalent to the terms "amplimer,""primer," "oligomer," and "probe," as these terms are commonly definedin the art.

"Peptide nucleic acid" (PNA), as used herein, refers to an antisensemolecule or anti-gene agent which comprises an oligonucleotide of atleast about 5 nucleotides in length linked to a peptide backbone ofamino acid residues ending in lysine. The terminal lysine conferssolubility to the composition. PNAs preferentially bind complementarysingle stranded DNA or RNA and stop transcript elongation, and may bepegylated to extend their lifespan in the cell. (See, e.g., Nielsen, P.E. et al. (1993) Anticancer Drug Des. 8:53-63.)

The term "sample," as used herein, is used in its broadest sense. Abiological sample suspected of containing nucleic acids encoding HITLP,or fragments thereof, or HITLP itself, may comprise a bodily fluid; anextract from a cell, chromosome, organelle, or membrane isolated from acell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a solidsupport; a tissue; a tissue print; etc.

As used herein, the terms "specific binding" or "specifically binding"refer to that interaction between a protein or peptide and an agonist,an antibody, or an antagonist. The interaction is dependent upon thepresence of a particular structure of the protein, e.g., the antigenicdeterminant or epitope, recognized by the binding molecule. For example,if an antibody is specific for epitope "A," the presence of apolypeptide containing the epitope A, or the presence of free unlabeledA, in a reaction containing free labeled A and the antibody will reducethe amount of labeled A that binds to the antibody.

As used herein, the term "stringent conditions" refers to conditionswhich permit hybridization between polynucleotides and the claimedpolynucleotides. Stringent conditions can be defined by saltconcentration, the concentration of organic solvent, e.g., formamide,temperature, and other conditions well known in the art. In particular,stringency can be increased by reducing the concentration of salt,increasing the concentration of formamide, or raising the hybridizationtemperature.

The term "substantially purified," as used herein, refers to nucleicacid or amino acid sequences that are removed from their naturalenvironment and are isolated or separated, and are at least about 60%free, preferably about 75% free, and most preferably about 90% free fromother components with which they are naturally associated.

A "substitution," as used herein, refers to the replacement of one ormore amino acids or nucleotides by different amino acids or nucleotides,respectively.

"Transformation," as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. Transformation mayoccur under natural or artificial conditions according to variousmethods well known in the art, and may rely on any known method for theinsertion of foreign nucleic acid sequences into a prokaryotic oreukaryotic host cell. The method for transformation is selected based onthe type of host cell being transformed and may include, but is notlimited to, viral infection, electroporation, heat shock, lipofection,and particle bombardment. The term "transformed" cells includes stablytransformed cells in which the inserted DNA is capable of replicationeither as an autonomously replicating plasmid or as part of the hostchromosome, as well as transiently transformed cells which express theinserted DNA or RNA for limited periods of time.

A "variant" of HITLP polypeptides, as used herein, refers to an aminoacid sequence that is altered by one or more amino acid residues. Thevariant may have "conservative" changes, wherein a substituted aminoacid has similar structural or chemical properties (e.g., replacement ofleucine with isoleucine). More rarely, a variant may have"nonconservative" changes (e.g., replacement of glycine withtryptophan). Analogous minor variations may also include amino aciddeletions or insertions, or both. Guidance in determining which aminoacid residues may be substituted, inserted, or deleted withoutabolishing biological or immunological activity may be found usingcomputer programs well known in the art, for example, LASERGENE™software.

The term "variant," when used in the context of a polynucleotidesequence, may encompass a polynucleotide sequence related to HITLP. Thisdefinition may also include, for example, "allelic" (as defined above),"splice," "species," or "polymorphic" variants. A splice variant mayhave significant identity to a reference molecule, but will generallyhave a greater or lesser number of polynucleotides due to alternatesplicing of exons during mRNA processing. The corresponding polypeptidemay possess additional functional domains or an absence of domains.Species variants are polynucleotide sequences that vary from one speciesto another. The resulting polypeptides generally will have significantamino acid identity relative to each other. A polymorphic variant is avariation in the polynucleotide sequence of a particular gene betweenindividuals of a given species. Polymorphic variants also may encompass"single nucleotide polymorphisms" (SNPs) in which the polynucleotidesequence varies by one base. The presence of SNPs may be indicative of,for example, a certain population, a disease state, or a propensity fora disease state.

THE INVENTION

The invention is based on the discovery of a new human iontransport-like protein (HITLP), the polynucleotides encoding HITLP, andthe use of these compositions for the diagnosis, treatment, orprevention of osmoregulatory and inflammatory disorders.

Nucleic acids encoding the HITLP of the present invention were firstidentified in Incyte Clone 3171334 from the breast tissue cDNA library(BRSTNOT18) using a computer search, e.g., BLAST, for amino acidsequence alignments. A consensus sequence, SEQ ID NO:2, was derived fromthe following overlapping and/or extended nucleic acid sequences: IncyteClones 3171334H1 and 3171334F7 (BRSTNOT18).

In one embodiment, the invention encompasses a polypeptide comprisingthe amino acid sequence of SEQ ID NO:1, as shown in FIGS. 1A and 1B.HITLP is 109 amino acids in length and has a potential amidation site atresidue L106; two potential N-glycosylation sites at residues N29 andN46; a potential protein kinase C phosphorylation site at residue S31;and a potential tyrosine kinase phosphorylation site at residue Y61.PRINTS identifies three crustacean hyperglycemic hormone signaturesequences from residues F36 through F50, N46 through Q62, and Q62through C77. SPScan identifies a potential signal sequence from residueM1 through A23. As shown in FIG. 2, HITLP has chemical and structuralsimilarity with locust ion transport protein (GI 1244522; SEQ ID NO:3)and locust ion transport-like protein (GI 1244524; SEQ ID NO:4). Inparticular, HITLP and locust ion transport protein share 41% identity;HITLP and locust ion transport-like protein share 39% identity; andHITLP, locust ion transport protein, and locust ion transport-likeprotein share a CHH-sequence consisting of a K-R cleavage site (residuesK32 and R33 in HITLP), six conserved cysteine residues at C41, C57, C60,C73, C77, and C86 in HITLP, and a potential C-terminal amidation site atresidue L106 in HITLP. A region of unique sequence in human iontransport-like protein from about amino acid 99 to about amino acid 105is encoded by a fragment of SEQ ID NO:2 from about nucleotide 364 toabout nucleotide 384. Northern analysis shows the expression of thissequence in a library derived from a human breast tissue (BRSTNOT18)which is associated with both cancer and an inflammatory response.

The invention also encompasses HITLP variants. A preferred HITLP variantis one which has at least about 80%, more preferably at least about 90%,and most preferably at least about 95% amino acid sequence identity tothe HITLP amino acid sequence, and which contains at least onefunctional or structural characteristic of HITLP.

The invention also encompasses polynucleotides which encode HITLP. In aparticular embodiment, the invention encompasses a polynucleotidesequence comprising the sequence of SEQ ID NO:2, which encodes an HITLP.

The invention also encompasses a variant of a polynucleotide sequenceencoding HITLP. In particular, such a variant polynucleotide sequencewill have at least about 70%, more preferably at least about 85%, andmost preferably at least about 95% polynucleotide sequence identity tothe polynucleotide sequence encoding HITLP. A particular aspect of theinvention encompasses a variant of SEQ ID NO:2 which has at least about70%, more preferably at least about 85%, and most preferably at leastabout 95% polynucleotide sequence identity to SEQ ID NO:2. Any one ofthe polynucleotide variants described above can encode an amino acidsequence which contains at least one functional or structuralcharacteristic of HITLP.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of polynucleotidesequences encoding HITLP, some bearing minimal similarity to thepolynucleotide sequences of any known and naturally occurring gene, maybe produced. Thus, the invention contemplates each and every possiblevariation of polynucleotide sequence that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code as applied tothe polynucleotide sequence of naturally occurring HITLP, and all suchvariations are to be considered as being specifically disclosed.

Although nucleotide sequences which encode HITLP and its variants arepreferably capable of hybridizing to the nucleotide sequence of thenaturally occurring HITLP under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding HITLP possessing a substantially different codon usage, e.g.,inclusion of non-naturally occurring codons. Codons may be selected toincrease the rate at which expression of the peptide occurs in aparticular prokaryotic or eukaryotic host in accordance with thefrequency with which particular codons are utilized by the host. Otherreasons for substantially altering the nucleotide sequence encodingHITLP and its derivatives without altering the encoded amino acidsequences include the production of RNA transcripts having moredesirable properties, such as a greater half-life, than transcriptsproduced from the naturally occurring sequence.

The invention also encompasses production of DNA sequences which encodeHITLP and HITLP derivatives, or fragments thereof, entirely by syntheticchemistry. After production, the synthetic sequence may be inserted intoany of the many available expression vectors and cell systems usingreagents well known in the art. Moreover, synthetic chemistry may beused to introduce mutations into a sequence encoding HITLP or anyfragment thereof.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridizing to the claimed polynucleotide sequences, and, inparticular, to those shown in SEQ ID NO:2, or a fragment of SEQ ID NO:2,under various conditions of stringency. (See, e.g., Wahl, G. M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A. R. (1987)Methods Enzymol. 152:507-511.) For example, stringent salt concentrationwill ordinarily be less than about 750 mM NaCl and 75 mM trisodiumcitrate, preferably less than about 500 mM NaCl and 50 mM trisodiumcitrate, and most preferably less than about 250 mM NaCl and 25 mMtrisodium citrate. Low stringency hybridization can be obtained in theabsence of organic solvent, e.g., formamide, while high stringencyhybridization can be obtained in the presence of at least about 35%formamide, and most preferably at least about 50% formamide. Stringenttemperature conditions will ordinarily include temperatures of at leastabout 30° C., more preferably of at least about 37° C., and mostpreferably of at least about 42° C. Varying additional parameters, suchas hybridization time, the concentration of detergent, e.g., sodiumdodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA,are well known to those skilled in the art. Various levels of stringencyare accomplished by combining these various conditions as needed. In apreferred embodiment, hybridization will occur at 30° C. in 750 mM NaCl,75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment,hybridization will occur at 37° C. in 500 mM NaCl, 50 mM trisodiumcitrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA(ssDNA). In a most preferred embodiment, hybridization will occur at 42°C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and200 μg/ml ssDNA. Useful variations on these conditions will be readilyapparent to those skilled in the art.

The washing steps which follow hybridization can also vary instringency. Wash stringency conditions can be defined by saltconcentration and by temperature. As above, wash stringency can beincreased by decreasing salt concentration or by increasing temperature.For example, stringent salt concentration for the wash steps willpreferably be less than about 30 mM NaCl and 3 mM trisodium citrate, andmost preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.Stringent temperature conditions for the wash steps will ordinarilyinclude temperature of at least about 25° C., more preferably of atleast about 42° C., and most preferably of at least about 68° C. In apreferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, washsteps will occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and0.1% SDS. In a most preferred embodiment, wash steps will occur at 68°C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additionalvariations on these conditions will be readily apparent to those skilledin the art.

Methods for DNA sequencing and analysis are well known in the art. Themethods may employ such enzymes as the Klenow fragment of DNA polymerase1, SEQUENASE® (Amersham Pharmacia Biotech Ltd., Uppsala, Sweden), Taqpolymerase (The Perkin-Elmer Corp., Norwalk, Conn.), thermostable T7polymerase (Amersham Pharmacia Biotech Ltd., Uppsala, Sweden), orcombinations of polymerases and proofreading exonucleases, such as thosefound in the ELONGASE™ amplification system (Life Technologies, Inc.,Rockville, Md.). Preferably, sequence preparation is automated withmachines, e.g., the ABI CATALYST™ 800 (The Perkin-Elmer Corp., Norwalk,Conn.) or MICROLAB® 2200 (Hamilton Co., Reno, Nev.) systems, incombination with thermal cyclers. Sequencing can also be automated, suchas by ABI PRISM™ 373 or 377 systems (The Perkin-Elmer Corp., Norwalk,Conn.) or the MEGABACE™ 1000 capillary electrophoresis system (MolecularDynamics, Inc., Sunnyvale, Calif.). Sequences can be analyzed usingcomputer programs and algorithms well known in the art. (See, e.g.,Ausubel, supra, unit 7.7; and Meyers, R. A. (1995) Molecular Biology andBiotechnology, Wiley VCH, Inc, New York, N.Y.)

The nucleic acid sequences encoding HITLP may be extended utilizing apartial nucleotide sequence and employing various PCR-based methodsknown in the art to detect upstream sequences, such as promoters andregulatory elements. For example, one method which may be employed,restriction-site PCR, uses universal and nested primers to amplifyunknown sequence from genomic DNA within a cloning vector. (See, e.g.,Sarkar, G. (1993) PCR Methods Applic. 2:318-322.) Another method,inverse PCR, uses primers that extend in divergent directions to amplifyunknown sequence from a circularized template. The template is derivedfrom restriction fragments comprising a known genomic locus andsurrounding sequences. (See, e.g., Triglia, T. et al. (1988) NucleicAcids Res. 16:8186.) A third method, capture PCR, involves PCRamplification of DNA fragments adjacent to known sequences in human andyeast artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al.(1991) PCR Methods Applic. 1:111-119.) In this method, multiplerestriction enzyme digestions and ligations may be used to insert anengineered double-stranded sequence into a region of unknown sequencebefore performing PCR. Other methods which may be used to retrieveunknown sequences are known in the art. (See, e.g., Parker, J. D. et al.(1991) Nucleic Acids Res. 19:3055-306). Additionally, one may use PCR,nested primers, and PromoterFinder™ libraries to walk genomic DNA(Clontech, Palo Alto, Calif.). This procedure avoids the need to screenlibraries and is useful in finding intron/exon junctions. For allPCR-based methods, primers may be designed using commercially availablesoftware, such as OLIGO™ 4.06 Primer Analysis software (NationalBiosciences Inc., Plymouth, Minn.) or another appropriate program, to beabout 22 to 30 nucleotides in length, to have a GC content of about 50%or more, and to anneal to the template at temperatures of about 68° C.to 72° C.

When screening for full-length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. In addition,random-primed libraries, which often include sequences containing the 5'regions of genes, are preferable for situations in which an oligo d(T)library does not yield a full-length cDNA. Genomic libraries may beuseful for extension of sequence into 5' non-transcribed regulatoryregions.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentnucleotide-specific, laser-stimulated fluorescent dyes, and a chargecoupled device camera for detection of the emitted wavelengths.Output/light intensity may be converted to electrical signal usingappropriate software (e.g., Genotyper™ and Sequence Navigator™, (ThePerkin-Elmer Corp., Norwalk, Conn.)), and the entire process fromloading of samples to computer analysis and electronic data display maybe computer controlled. Capillary electrophoresis is especiallypreferable for sequencing small DNA fragments which may be present inlimited amounts in a particular sample.

In another embodiment of the invention, polynucleotide sequences orfragments thereof which encode HITLP may be cloned in recombinant DNAmolecules that direct expression of HITLP, or fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced and used to express HITLP.

The nucleotide sequences of the present invention can be engineeredusing methods generally known in the art in order to alterHITLP-encoding sequences for a variety of purposes including, but notlimited to, modification of the cloning, processing, and/or expressionof the gene product. DNA shuffling by random fragmentation and PCRreassembly of gene fragments and synthetic oligonucleotides may be usedto engineer the nucleotide sequences. For example,oligonucleotide-mediated site-directed mutagenesis may be used tointroduce mutations that create new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, and so forth.

In another embodiment, sequences encoding HITLP may be synthesized, inwhole or in part, using chemical methods well known in the art. (See,e.g., Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser.215-223, and Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser.225-232.) Alternatively, HITLP itself or a fragment thereof may besynthesized using chemical methods. For example, peptide synthesis canbe performed using various solid-phase techniques. (See, e.g., Roberge,J. Y. et al. (1995) Science 269:202-204.) Automated synthesis may beachieved using the ABI 431A Peptide Synthesizer (The Perkin-Elmer Corp.,Norwalk, Conn.). Additionally, the amino acid sequence of HITLP, or anypart thereof, may be altered during direct synthesis and/or combinedwith sequences from other proteins, or any part thereof, to produce avariant polypeptide.

The peptide may be substantially purified by preparative highperformance liquid chromatography. (See, e.g, Chiez, R. M. and F. Z.Regnier (1990) Methods Enzymol. 182:392-421.) The composition of thesynthetic peptides may be confirmed by amino acid analysis or bysequencing. (See, e.g., Creighton, T. (1984) Proteins, Structures andMolecular Properties, WH Freeman and Co., New York, N.Y.)

In order to express a biologically active HITLP, the nucleotidesequences encoding HITLP or derivatives thereof may be inserted into anappropriate expression vector, i.e., a vector which contains thenecessary elements for transcriptional and translational control of theinserted coding sequence in a suitable host. These elements includeregulatory sequences, such as enhancers, constitutive and induciblepromoters, and 5' and 3' untranslated regions in the vector and inpolynucleotide sequences encoding HITLP. Such elements may vary in theirstrength and specificity. Specific initiation signals may also be usedto achieve more efficient translation of sequences encoding HITLP. Suchsignals include the ATG initiation codon and adjacent sequences, e.g.the Kozak sequence. In cases where sequences encoding HITLP and itsinitiation codon and upstream regulatory sequences are inserted into theappropriate expression vector, no additional transcriptional ortranslational control signals may be needed. However, in cases whereonly coding sequence, or a fragment thereof, is inserted, exogenoustranslational control signals including an in-frame ATG initiation codonshould be provided by the vector. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers appropriate for the particular host cell system used. (See,e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.)

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing sequences encoding HITLP andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. (See, e.g., Sambrook, J.et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring HarborPress, Plainview, N.Y., ch. 4, 8, and 16-17; and Ausubel, F. M. et al.(1995, and periodic supplements) Current Protocols in Molecular Biology,John Wiley & Sons, New York, N.Y., ch. 9, 13, and 16.)

A variety of expression vector/host systems may be utilized to containand express sequences encoding HITLP. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith viral expression vectors (e.g., baculovirus); plant cell systemstransformed with viral expression vectors (e.g., cauliflower mosaicvirus (CaMV) or tobacco mosaic virus (TMV)) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Theinvention is not limited by the host cell employed.

In bacterial systems, a number of cloning and expression vectors may beselected depending upon the use intended for polynucleotide sequencesencoding HITLP. For example, routine cloning, subcloning, andpropagation of polynucleotide sequences encoding HITLP can be achievedusing a multifunctional E. coli vector such as Bluescript® (Stratagene)or pSport1™ plasmid (GIBCO BRL). Ligation of sequences encoding HITLPinto the vector's multiple cloning site disrupts the lacZ gene, allowinga colorimetric screening procedure for identification of transformedbacteria containing recombinant molecules. In addition, these vectorsmay be useful for in vitro transcription, dideoxy sequencing, singlestrand rescue with helper phage, and creation of nested deletions in thecloned sequence. (See, e.g., Van Heeke, G. and S. M. Schuster (1989) J.Biol. Chem. 264:5503-5509.) When large quantities of HITLP are needed,e.g. for the production of antibodies, vectors which direct high levelexpression of HITLP may be used. For example, vectors containing thestrong, inducible T5 or T7 bacteriophage promoter may be used.

Yeast expression systems may be used for production of HITLP. A numberof vectors containing constitutive or inducible promoters, such as alphafactor, alcohol oxidase, and PGH, may be used in the yeast Saccharomycescerevisiae or Pichia pastoris. In addition, such vectors direct eitherthe secretion or intracellular retention of expressed proteins andenable integration of foreign sequences into the host genome for stablepropagation. (See, e.g., Ausubel, supra; and Grant et al. (1987) MethodsEnzymol. 153:516-54; Scorer, C. A. et al. (1994) Bioffechnology12:181-184.)

Plant systems may also be used for expression of HITLP. Transcription ofsequences encoding HITLP may be driven viral promoters, e.g., the 35Sand 19S promoters of CaMV used alone or in combination with the omegaleader sequence from TMV. (Takamatsu, N. (1987) EMBO J. 6:307-311.)Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984)EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; andWinter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105.) Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. (See, e.g., Hobbs, S.or Murry, L. E. in McGraw Hill Yearbook of Science and Technology (1992)McGraw Hill, New York, N.Y.; pp. 191-196.)

In mammalian cells, a number of viral-based expression systems may beutilized. In cases where an adenovirus is used as an expression vector,sequences encoding HITLP may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain infective virus whichexpresses HITLP in host cells. (See, e.g., Logan, J. and T. Shenk (1984)Proc. Natl. Acad. Sci. 81:3655-3659.) In addition, transcriptionenhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used toincrease expression in mammalian host cells. SV40 or EBV-based vectorsmay also be used for high-level protein expression.

Human artificial chromosomes (HACs) may also be employed to deliverlarger fragments of DNA than can be contained in and expressed from aplasmid. HACs of about 6 kb to 10 Mb are constructed and delivered viaconventional delivery methods (liposomes, polycationic amino polymers,or vesicles) for therapeutic purposes.

For long term production of recombinant proteins in mammalian systems,stable expression of HITLP in cell lines is preferred. For example,sequences encoding HITLP can be transformed into cell lines usingexpression vectors which may contain viral origins of replication and/orendogenous expression elements and a selectable marker gene on the sameor on a separate vector. Following the introduction of the vector, cellsmay be allowed to grow for about 1 to 2 days in enriched media beforebeing switched to selective media. The purpose of the selectable markeris to confer resistance to a selective agent, and its presence allowsgrowth and recovery of cells which successfully express the introducedsequences. Resistant clones of stably transformed cells may bepropagated using tissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase and adenine phosphoribosyltransferase genes, for use intk⁻ or apr⁻ cells, respectively. (See, e.g., Wigler, M. et al. (1977)Cell 11:223-232; and Lowy, I. et al. (1980) Cell 22:817-823.) Also,antimetabolite, antibiotic, or herbicide resistance can be used as thebasis for selection. For example, dhfr confers resistance tomethotrexate; neo confers resistance to the aminoglycosides neomycin andG-418; and ais or pat confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively. (See, e.g., Wigler, M.et al. (1980) Proc. Natl. Acad. Sci. 77:3567-3570; Colbere-Garapin, F.et al (1981) J. Mol. Biol. 150:1-14; and Murry, supra.) Additionalselectable genes have been described, e.g., trpB and hisD, which altercellular requirements for metabolites. (See, e.g., Hartman, S. C. and R.C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-8051.) Visiblemarkers, e.g., anthocyanins, green fluorescent proteins (GFP) (Clontech,Palo Alto, Calif.), β glucuronidase and its substrate β-D-glucuronoside,or luciferase and its substrate luciferin may be used. These markers canbe used not only to identify transformants, but also to quantify theamount of transient or stable protein expression attributable to aspecific vector system. (See, e.g., Rhodes, C. A. et al. (1995) MethodsMol. Biol. 55:121-131.)

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, the presence and expression of thegene may need to be confirmed. For example, if the sequence encodingHITLP is inserted within a marker gene sequence, transformed cellscontaining sequences encoding HITLP can be identified by the absence ofmarker gene function. Alternatively, a marker gene can be placed intandem with a sequence encoding HITLP under the control of a singlepromoter. Expression of the marker gene in response to induction orselection usually indicates expression of the tandem gene as well.

In general, host cells that contain the nucleic acid sequence encodingHITLP and that express HITLP may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCRamplification, and protein bioassay or immunoassay techniques whichinclude membrane, solution, or chip based technologies for the detectionand/or quantification of nucleic acid or protein sequences.

Immunological methods for detecting and measuring the expression ofHITLP using either specific polyclonal or monoclonal antibodies areknown in the art. Examples of such techniques include enzyme-linkedimmunosorbent assays (ELISAs), radioimmunoassays (RIAs), andfluorescence activated cell sorting (FACS). A two-site, monoclonal-basedimmunoassay utilizing monoclonal antibodies reactive to twonon-interfering epitopes on HITLP is preferred, but a competitivebinding assay may be employed. These and other assays are well known inthe art. (See, e.g., Hampton, R. et al. (1990) Serological Methods, aLaboratory Manual, APS Press, St Paul, Minn., Section IV; Coligan, J. E.et al. (1997 and periodic supplements) Current Protocols in Immunology,Greene Pub. Associates and Wiley-Interscience, New York, N.Y.; andMaddox, D. E. et al. (1983) J. Exp. Med. 158:1211-1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding HITLP includeoligolabeling, nick translation, end-labeling, or PCR amplificationusing a labeled nucleotide. Alternatively, the sequences encoding HITLP,or any fragments thereof, may be cloned into a vector for the productionof an mRNA probe. Such vectors are known in the art, are commerciallyavailable, and may be used to synthesize RNA probes in vitro by additionof an appropriate RNA polymerase such as T7, T3, or SP6 and labelednucleotides. These procedures may be conducted using a variety ofcommercially available kits, such as those provided by Pharmacia &Upjohn (Kalamazoo, Mich.), Promega (Madison, Wis.), and U.S. BiochemicalCorp. (Cleveland, Ohio). Suitable reporter molecules or labels which maybe used for ease of detection include radionuclides, enzymes,fluorescent, chemiluminescent, or chromogenic agents, as well assubstrates, cofactors, inhibitors, magnetic particles, and the like.

Host cells transformed with nucleotide sequences encoding HITLP may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a transformedcell may be secreted or retained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodeHITLP may be designed to contain signal sequences which direct secretionof HITLP through a prokaryotic or eukaryotic cell membrane.

In addition, a host cell strain may be chosen for its ability tomodulate expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a "prepro" form of theprotein may also be used to specify protein targeting, folding, and/oractivity. Different host cells which have specific cellular machineryand characteristic mechanisms for post-translational activities (e.g.,CHO, HeLa, MDCK, HEK293, and W138), are available from the American TypeCulture Collection (ATCC, Bethesda, Md.) and may be chosen to ensure thecorrect modification and processing of the foreign protein.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding HITLP may be ligated to aheterologous sequence resulting in translation of a fusion protein inany of the aforementioned host systems. For example, a chimeric HITLPprotein containing a heterologous moiety that can be recognized by acommercially available antibody may facilitate the screening of peptidelibraries for inhibitors of HITLP activity. Heterologous protein andpeptide moieties may also facilitate purification of fusion proteinsusing commercially available affinity matrices. Such moieties include,but are not limited to, glutathione S-transferase (GST), maltose bindingprotein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP),6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and6-His enable purification of their cognate fusion proteins onimmobilized glutathione, maltose, phenylarsine oxide, calmodulin, andmetal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA)enable immunoaffinity purification of fusion proteins using commerciallyavailable monoclonal and polyclonal antibodies that specificallyrecognize these epitope tags. A fusion protein may also be engineered tocontain a proteolytic cleavage site located between the HITLP encodingsequence and the heterologous protein sequence, so that HITLP may becleaved away from the heterologous moiety following purification.Methods for fusion protein expression and purification are discussed inAusubel, F. M. et al. (1995 and periodic supplements) Current Protocolsin Molecular Biology, John Wiley & Sons, New York, N.Y., ch 10. Avariety of commercially available kits may also be used to facilitateexpression and purification of fusion proteins.

In a further embodiment of the invention, synthesis of radiolabeledHITLP may be achieved in vitro using the TNT™ rabbit reticulocyte lysateor wheat germ extract systems (Promega, Madison, Wis.). These systemscouple transcription and translation of protein-coding sequencesoperably associated with the T7, T3, or SP6 promoters. Translation takesplace in the presence of a radiolabeled amino acid precursor, preferably³⁵ S-methionine.

Fragments of HITLP may be produced not only by recombinant production,but also by direct peptide synthesis using solid-phase techniques. (See,e.g., Creighton, supra pp. 55-60.) Protein synthesis may be performed bymanual techniques or by automation. Automated synthesis may be achieved,for example, using the Applied Biosystems 431 A Peptide Synthesizer (ThePerkin-Elmer Corp., Norwalk, Conn.). Various fragments of HITLP may besynthesized separately and then combined to produce the full lengthmolecule.

THERAPEUTICS

Chemical and structural similarity, e.g., in the context of sequencesand motifs, exists among HITLP, locust ion transport-like protein (GI1244522) and locust ion transport protein (GI 1244524). In addition,HITLP is expressed in breast tissue associated with cancer andinflammation. Therefore, HITLP appears to play a role in osmoregulatoryand inflammatory disorders.

Therefore, in one embodiment, HITLP or a fragment or derivative thereofmay be administered to a subject to treat or prevent an osmoregulatorydisorder. Such osmoregulatory disorders can include, but are not limitedto, diabetes insipidus, diarrhea, peritonitis, chronic renal failure,Addison's disease, syndrome of inappropriate antidiuretic hormone(SIADH), hypoaldosteronism, hyponatremia, adrenal insufficiency,hypothyroidism, hypernatremia, hypokalemia, Barter's syndrome, Cushing'ssyndrome, metabolic acidosis, metabolic alkalosis, encephalopathy,edema, hypotension, and hypertension.

In another embodiment, a vector capable of expressing HITLP or afragment or derivative thereof may be administered to a subject to treator prevent an osmoregulatory disorder including, but not limited to,those described above.

In a further embodiment, a pharmaceutical composition comprising asubstantially purified HITLP in conjunction with a suitablepharmaceutical carrier may be administered to a subject to treat orprevent an osmoregulatory disorder including, but not limited to, thoseprovided above.

In still another embodiment, an agonist which modulates the activity ofHITLP may be administered to a subject to treat or prevent anosmoregulatory disorder including, but not limited to, those listedabove.

In another embodiment, HITLP or a fragment or derivative thereof may beadministered to a subject to treat or prevent an inflammatory disorder.Such inflammatory disorders can include, but are not limited to,acquired immunodeficiency syndrome (AIDS), adult respiratory distresssyndrome, allergies, ankylosing spondylitis, amyloidosis, anemia,asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmunethyroiditis, bronchitis, cholecystitis, contact dermatitis, Crohn'sdisease, atopic dermatitis, dermatomyositis, diabetes mellitus,emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosisfetalis, erythema nodosum, atrophic gastritis, glomerulonephritis,Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis,hypereosinophilia, irritable bowel syndrome, multiple sclerosis,myasthenia gravis, myocardial or pericardial inflammation,osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis,Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren'ssyndrome, systemic anaphylaxis, systemic lupus erythematosus, systemicsclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Wernersyndrome, complications of cancer, hemodialysis, and extracorporealcirculation, viral, bacterial, fungal, parasitic, protozoal, andhelminthic infections, and trauma.

In another embodiment, a vector capable of expressing HITLP or afragment or derivative thereof may be administered to a subject to treator prevent an inflammatory disorder including, but not limited to, thosedescribed above.

In a further embodiment, a pharmaceutical composition comprising asubstantially purified HITLP in conjunction with a suitablepharmaceutical carrier may be administered to a subject to treat orprevent an inflammatory disorder including, but not limited to, thoseprovided above.

In still another embodiment, an agonist which modulates the activity ofHITLP may be administered to a subject to treat or prevent aninflammatory disorder including, but not limited to, those listed above.

In other embodiments, any of the proteins, antagonists, antibodies,agonists, complementary sequences, or vectors of the invention may beadministered in combination with other appropriate therapeutic agents.Selection of the appropriate agents for use in combination therapy maybe made by one of ordinary skill in the art, according to conventionalpharmaceutical principles. The combination of therapeutic agents may actsynergistically to effect the treatment or prevention of the variousdisorders described above. Using this approach, one may be able toachieve therapeutic efficacy with lower dosages of each agent, thusreducing the potential for adverse side effects.

An antagonist of HITLP may be produced using methods which are generallyknown in the art. In particular, purified HITLP may be used to produceantibodies or to screen libraries of pharmaceutical agents to identifythose which specifically bind HITLP. Antibodies to HITLP may also begenerated using methods that are well known in the art. Such antibodiesmay include, but are not limited to, polyclonal, monoclonal, chimeric,and single chain antibodies, Fab fragments, and fragments produced by aFab expression library. Neutralizing antibodies (i.e., those whichinhibit dimer formation) are especially preferred for therapeutic use.

For the production of polyclonal antibodies, various hosts includinggoats, rabbits, rats, mice, humans, and others may be immunized byinjection with HITLP or with any fragment or oligopeptide thereof whichhas immunogenic properties. Rats and mice are preferred hosts fordownstream applications involving monoclonal antibody production.Depending on the host species, various adjuvants may be used to increaseimmunological response. Such adjuvants include, but are not limited to,Freund's, mineral gels such as aluminum hydroxide, and surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, KLH, and dinitrophenol. Among adjuvants used in humans,BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are especiallypreferable. (For review of methods for antibody production and analysis,see, e.g., Harlow, E. and Lane, D. (1988) Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.)

It is preferred that the oligopeptides, peptides, or fragments used toinduce antibodies to HITLP have an amino acid sequence consisting of atleast about 5 amino acids, and, more preferably, of at least about 14amino acids. It is also preferable that these oligopeptides, peptides,or fragments are identical to a portion of the amino acid sequence ofthe natural protein and contain the entire amino acid sequence of asmall, naturally occurring molecule. Short stretches of HITLP aminoacids may be fused with those of another protein, such as KLH, andantibodies to the chimeric molecule may be produced.

Monoclonal antibodies to HITLP may be prepared using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the EBV-hybridomatechnique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497;Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. etal. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; and Cole, S.P. et al.(1984) Mol. Cell Biol. 62:109-120.)

In addition, techniques developed for the production of "chimericantibodies," such as the splicing of mouse antibody genes to humanantibody genes to obtain a molecule with appropriate antigen specificityand biological activity, can be used. (See, e.g., Morrison, S. L. et al.(1984) Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al.(1984) Nature 312:604-608; and Takeda, S. et al. (1985) Nature314:452-454.) Alternatively, techniques described for the production ofsingle chain antibodies may be adapted, using methods known in the art,to produce HITLP-specific single chain antibodies. Antibodies withrelated specificity, but of distinct idiotypic composition, may begenerated by chain shuffling from random combinatorial immunoglobulinlibraries. (See, e.g., Burton D. R. (1991) Proc. Natl. Acad. Sci.88:10134-10137.)

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature.(See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837; and Winter, G. et al. (1991) Nature 349:293-299.)

Antibody fragments which contain specific binding sites for HITLP mayalso be generated. For example, such fragments include, but are notlimited to, F(ab')2 fragments produced by pepsin digestion of theantibody molecule and Fab fragments generated by reducing the disulfidebridges of the F(ab')2 fragments. Alternatively, Fab expressionlibraries may be constructed to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity. (See, e.g., Huse,W. D. et al. (1989) Science 246:1275-1281.)

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity and minimal cross-reactivity. Numerousprotocols for competitive binding or immunoradiometric assays usingeither polyclonal or monoclonal antibodies with establishedspecificities are well known in the art. Such immunoassays typicallyinvolve the measurement of complex formation between HITLP and itsspecific antibody. A two-site, monoclonal-based immunoassay utilizingmonoclonal antibodies reactive to two non-interfering HITLP epitopes ispreferred, but a competitive binding assay may also be employed.(Maddox, supra.)

Various methods such as Scatchard analysis in conjunction withradioimmunoassay techniques may be used to assess the affinity ofantibodies for HITLP. Affinity is expressed as an association constant,K_(a), which is defined as the molar concentration of HITLP-antibodycomplex divided by the molar concentrations of free antigen and freeantibody under equilibrium conditions. The K_(a) determined for apreparation of polyclonal antibodies, which are heterogeneous in theiraffinities for multiple HITLP epitopes, represents the average affinity,or avidity, of the antibodies for HITLP. The K_(a) determined for apreparation of monoclonal antibodies, which are monospecific for aparticular HITLP epitope, represents a true measure of affinity.High-affinity antibody preparations with K_(a) ranging from about 10⁹ to10¹² L/mole are preferred for use in immunoassays in which theHITLP-antibody complex must withstand rigorous manipulations.Low-affinity antibody preparations with K_(a) ranging from about 10⁶ to10⁷ L/mole are preferred for use in immunopurification and similarprocedures which ultimately require dissociation of HITLP, preferably inactive form, from the antibody. (Catty, D. (1988) Antibodies. Volume I:A Practical Approach, IRL Press, Washington, D. C.; and Liddell, J. E.and Cryer, A. (1991) A Practical Guide to Monoclonal Antibodies, JohnWiley & Sons, New York, N.Y.)

The titre and avidity of polyclonal antibody preparations may be furtherevaluated to determine the quality and suitability of such preparationsfor certain downstream applications. For example, a polyclonal antibodypreparation containing at least 1-2 mg specific antibody/ml, preferably5-10 mg specific antibody/ml, is preferred for use in proceduresrequiring precipitation of HITLP-antibody complexes. Procedures forevaluating antibody specificity, titer, and avidity, and guidelines forantibody quality and usage in various applications, are generallyavailable. (See, e.g., Catty, supra, and Coligan et al. supra.)

In another embodiment of the invention, the polynucleotides encodingHITLP, or any fragment or complement thereof, may be used fortherapeutic purposes. In one aspect, the complement of thepolynucleotide encoding HITLP may be used in situations in which itwould be desirable to block the transcription of the mRNA. Inparticular, cells may be transformed with sequences complementary topolynucleotides encoding HITLP. Thus, complementary molecules orfragments may be used to modulate HITLP activity, or to achieveregulation of gene function. Such technology is now well known in theart, and sense or antisense oligonucleotides or larger fragments can bedesigned from various locations along the coding or control regions ofsequences encoding HITLP.

Expression vectors derived from retroviruses, adenoviruses, or herpes orvaccinia viruses, or from various bacterial plasmids, may be used fordelivery of nucleotide sequences to the targeted organ, tissue, or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct vectors to express nucleic acid sequencescomplementary to the polynucleotides encoding HITLP. (See, e.g.,Sambrook, supra; and Ausubel, supra.)

Genes encoding HITLP can be turned off by transforming a cell or tissuewith expression vectors which express high levels of a polynucleotide,or fragment thereof, encoding HITLP. Such constructs may be used tointroduce untranslatable sense or antisense sequences into a cell. Evenin the absence of integration into the DNA, such vectors may continue totranscribe RNA molecules until they are disabled by endogenousnucleases. Transient expression may last for a month or more with anon-replicating vector, and may last even longer if appropriatereplication elements are part of the vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning complementary sequences or antisense molecules (DNA, RNA, orPNA) to the control, 5', or regulatory regions of the gene encodingHITLP. Oligonucleotides derived from the transcription initiation site,e.g., between about positions -10 and +10 from the start site, arepreferred. Similarly, inhibition can be achieved using triple helixbase-pairing methodology. Triple helix pairing is useful because itcauses inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature. (See, e.g., Gee, J. E. et al. (1994)in Huber, B. E. and B. I. Carr, Molecular and Immunologic Approaches,Futura Publishing Co., Mt. Kisco, N.Y., pp. 163-177.) A complementarysequence or antisense molecule may also be designed to block translationof mRNA by preventing the transcript from binding to ribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Forexample, engineered hammerhead motif ribozyme molecules may specificallyand efficiently catalyze endonucleolytic cleavage of sequences encodingHITLP.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites, including the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides, corresponding to the region of the target genecontaining the cleavage site, may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Complementary ribonucleic acid molecules and ribozymes of the inventionmay be prepared by any method known in the art for the synthesis ofnucleic acid molecules. These include techniques for chemicallysynthesizing oligonucleotides such as solid phase phosphoramiditechemical synthesis. Alternatively, RNA molecules may be generated by invitro and in vivo transcription of DNA sequences encoding HITLP. SuchDNA sequences may be incorporated into a wide variety of vectors withsuitable RNA polymerase promoters such as T7 or SP6. Alternatively,these cDNA constructs that synthesize complementary RNA, constitutivelyor inducibly, can be introduced into cell lines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5' and/or 3' ends of the molecule,or the use of phosphorothioate or 2'O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection, by liposome injections, or bypolycationic amino polymers may be achieved using methods which are wellknown in the art. (See, e.g., Goldman, C. K. et al. (1997) NatureBiotechnology 15:462-466.)

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such asdogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administrationof a pharmaceutical or sterile composition, in conjunction with apharmaceutically acceptable carrier, for any of the therapeutic effectsdiscussed above. Such pharmaceutical compositions may consist of HITLP,antibodies to HITLP, and mimetics, agonists, antagonists, or inhibitorsof HITLP. The compositions may be administered alone or in combinationwith at least one other agent, such as a stabilizing compound, which maybe administered in any sterile, biocompatible pharmaceutical carrierincluding, but not limited to, saline, buffered saline, dextrose, andwater. The compositions may be administered to a patient alone, or incombination with other agents, drugs, or hormones.

The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombining active compounds with solid excipient and processing theresultant mixture of granules (optionally, after grinding) to obtaintablets or dragee cores. Suitable auxiliaries can be added, if desired.Suitable excipients include carbohydrate or protein fillers, such assugars, including lactose, sucrose, mannitol, and sorbitol; starch fromcorn, wheat, rice, potato, or other plants; cellulose, such as methylcellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums, including arabic and tragacanth; andproteins, such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, and alginic acid or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with fillers or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils, such as sesame oil, or synthetic fatty acid esters, such asethyl oleate, triglycerides, or liposomes. Non-lipid polycationic aminopolymers may also be used for delivery. Optionally, the suspension mayalso contain suitable stabilizers or agents to increase the solubilityof the compounds and allow for the preparation of highly concentratedsolutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, and succinic acid. Salts tendto be more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1 mM to 50 mM histidine, 0.1% to 2% sucrose, and 2% to 7%mannitol, at a pH range of 4.5 to 5.5, that is combined with bufferprior to use.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. For administration of HITLP, such labeling would includeamount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells or inanimal models such as mice, rats, rabbits, dogs, or pigs. An animalmodel may also be used to determine the appropriate concentration rangeand route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example HITLP or fragments thereof, antibodies of HITLP,and agonists, antagonists or inhibitors of HITLP, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orwith experimental animals, such as by. calculating the ED₅₀ (the dosetherapeutically effective in 50% of the population) or LD₅₀ (the doselethal to 50% of the population) statistics. The dose ratio of toxic totherapuetic effects is the therapeutic index, and it can be expressed asthe LD₅₀ /ED₅₀ ratio. Pharmaceutical compositions which exhibit largetherapeutic indices are preferred. The data obtained from cell cultureassays and animal studies are used to formulate a range of dosage forhuman use. The dosage contained in such compositions is preferablywithin a range of circulating concentrations that includes the ED₅₀ withlittle or no toxicity. The dosage varies within this range dependingupon the dosage form employed, the sensitivity of the patient, and theroute of administration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject requiring treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, the generalhealth of the subject, the age, weight, and gender of the subject, timeand frequency of administration, drug combination(s), reactionsensitivities, and response to therapy. Longacting pharmaceuticalcompositions may be administered every 3 to 4 days, every week, orbiweekly depending on the half-life and clearance rate of the particularformulation.

Normal dosage amounts may vary from about 0.1 μg to 100,000 μg, up to atotal dose of about 1 gram, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

DIAGNOSTICS

In another embodiment, antibodies which specifically bind HITLP may beused for the diagnosis of disorders characterized by expression ofHITLP, or in assays to monitor patients being treated with HITLP oragonists, antagonists, or inhibitors of HITLP. Antibodies useful fordiagnostic purposes may be prepared in the same manner as describedabove for therapeutics. Diagnostic assays for HITLP include methodswhich utilize the antibody and a label to detect HITLP in human bodyfluids or in extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by covalent ornon-covalent attachment of a reporter molecule. A wide variety ofreporter molecules, several of which are described above, are known inthe art and may be used.

A variety of protocols for measuring HITLP, including ELISAs, RIAs, andFACS, are known in the art and provide a basis for diagnosing altered orabnormal levels of HITLP expression. Normal or standard values for HITLPexpression are established by combining body fluids or cell extractstaken from normal mammalian subjects, preferably human, with antibody toHITLP under conditions suitable for complex formation. The amount ofstandard complex formation may be quantitated by various methods,preferably by photometric means. Quantities of HITLP expressed insubject, control, and disease samples from biopsied tissues are comparedwith the standard values. Deviation between standard and subject valuesestablishes the parameters for diagnosing disease.

In another embodiment of the invention, the polynucleotides encodingHITLP may be used for diagnostic purposes. The polynucleotides which maybe used include oligonucleotide sequences, complementary RNA and DNAmolecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofHITLP may be correlated with disease. The diagnostic assay may be usedto determine absence, presence, and excess expression of HITLP, and tomonitor regulation of HITLP levels during therapeutic intervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding HITLP or closely related molecules may be used to identifynucleic acid sequences which encode HITLP. The specificity of the probe,whether it is made from a highly specific region, e.g., the 5'regulatory region, or from a less specific region, e.g., a conservedmotif, and the stringency of the hybridization or amplification(maximal, high, intermediate, or low), will determine whether the probeidentifies only naturally occurring sequences encoding HITLP, allelicvariants, or related sequences.

Probes may also be used for the detection of related sequences, andshould preferably have at least 50% sequence identity to any of theHITLP encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and may be derived from the sequence of SEQID NO:2 or from genomic sequences including promoters, enhancers, andintrons of the HITLP gene.

Means for producing specific hybridization probes for DNAs encodingHITLP include the cloning of polynucleotide sequences encoding HITLP orHITLP derivatives into vectors for the production of mRNA probes. Suchvectors are known in the art, are commercially available, and may beused to synthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, by radionuclides such as ³² p or ³⁵ S, or by enzymatic labels,such as alkaline phosphatase coupled to the probe via avidin/biotincoupling systems, and the like.

Polynucleotide sequences encoding HITLP may be used for the diagnosis ofa disorder associated with expression of HITLP. Examples of such adisorder include, but are not limited to, osmoregulatory disorders suchas diabetes insipidus, diarrhea, peritonitis, chronic renal failure,Addison's disease, SIADH, hypoaldosteronism, hyponatremia, adrenalinsufficiency, hypothyroidism, hypernatremia, hypokalemia, Barter'ssyndrome, Cushing's syndrome, metabolic acidosis, metabolic alkalosis,encephalopathy, edema, hypotension, and hypertension; and inflammatorydisorders such as acquired immunodeficiency syndrome (AIDS), adultrespiratory distress syndrome, allergies, ankylosing spondylitis,amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolyticanemia, autoimmune thyroiditis, bronchitis, cholecystitis, contactdermatitis, Crohn's disease, atopic dermatitis, dermatomyositis,diabetes mellitus, emphysema, episodic lymphopenia withlymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophicgastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves'disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowelsyndrome, multiple sclerosis, myasthenia gravis, myocardial orpericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis,scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupuserythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerativecolitis, uveitis, Werner syndrome, complications of cancer,hemodialysis, and extracorporeal circulation, viral, bacterial, fungal,parasitic, protozoal, and helminthic infections, and trauma. Thepolynucleotide sequences encoding HITLP may be used in Southern orNorthern analysis, dot blot, or other membrane-based technologies; inPCR technologies; in dipstick, pin, and ELISA assays; and in microarraysutilizing fluids or tissues from patients to detect altered HITLPexpression. Such qualitative or quantitative methods are well known inthe art.

In a particular aspect, the nucleotide sequences encoding HITLP may beuseful in assays that detect the presence of associated disorders,particularly those mentioned above. The nucleotide sequences encodingHITLP may be labeled by standard methods and added to a fluid or tissuesample from a patient under conditions suitable for the formation ofhybridization complexes. After a suitable incubation period, the sampleis washed and the signal is quantitated and compared with a standardvalue. If the amount of signal in the patient sample is significantlyaltered in comparison to a control sample then the presence of alteredlevels of nucleotide sequences encoding HITLP in the sample indicatesthe presence of the associated disorder. Such assays may also be used toevaluate the efficacy of a particular therapeutic treatment regimen inanimal studies, in clinical trials, or to monitor the treatment of anindividual patient.

In order to provide a basis for the diagnosis of a disorder associatedwith expression of HITLP, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, encoding HITLP, under conditionssuitable for hybridization or amplification. Standard hybridization maybe quantified by comparing the values obtained from normal subjects withvalues from an experiment in which a known amount of a substantiallypurified polynucleotide is used. Standard values obtained in this mannermay be compared with values obtained from samples from patients who aresymptomatic for a disorder. Deviation from standard values is used toestablish the presence of a disorder.

Once the presence of a disorder is established and a treatment protocolis initiated, hybridization assays may be repeated on a regular basis todetermine if the level of expression in the patient begins toapproximate that which is observed in the normal subject. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding HITLP may involve the use of PCR. These oligomers maybe chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably contain a fragment of a polynucleotideencoding HITLP, or a fragment of a polynucleotide complementary to thepolynucleotide encoding HITLP, and will be employed under optimizedconditions for identification of a specific gene or condition. Oligomersmay also be employed under less stringent conditions for detection orquantitation of closely related DNA or RNA sequences.

Methods which may also be used to quantitate the expression of HITLPinclude radiolabeling or biotinylating nucleotides, coamplification of acontrol nucleic acid, and interpolating results from standard curves.(See, e.g., Melby, P. C. et al. (1993) J. Immunol. Methods 159:235-244;and Duplaa, C. et al. (1993) Anal. Biochem. 229-236.) The speed ofquantitation of multiple samples may be accelerated by running the assayin an ELISA format where the oligomer of interest is presented invarious dilutions and a spectrophotometric or colorimetric responsegives rapid quantitation.

In further embodiments, oligonucleotides or longer fragments derivedfrom any of the polynucleotide sequences described herein may be used astargets in a microarray. The microarray can be used to monitor theexpression level of large numbers of genes simultaneously and toidentify genetic variants, mutations, and polymorphisms. Thisinformation may be used to determine gene function, to understand thegenetic basis of a disorder, to diagnose a disorder, and to develop andmonitor the activities of therapeutic agents.

Microarrays may be prepared, used, and analyzed using methods known inthe art. (See, e.g., Brennan, T. M. et al. (1995) U.S. Pat. No.5,474,796; Schena, M. et al. (1996) Proc. Nat. Acad. Sci.93:10614-10619; Baldeschweiler et al. (1995) PCT applicationW095/251116; Shalon, D. et al. (1995) PCT application WO95/35505;Heller, R. A. et al. (1997) Proc. Natl. Acad. Sci. 94:2150-2155; andHeller, M. J. et al. (1997) U.S. Pat. No. 5,605,662.)

In another embodiment of the invention, nucleic acid sequences encodingHITLP may be used to generate hybridization probes useful in mapping thenaturally occurring genomic sequence. The sequences may be mapped to aparticular chromosome, to a specific region of a chromosome, or toartificial chromosome constructions, e.g., human artificial chromosomes(HACs), yeast artificial chromosomes (YACs), bacterial artificialchromosomes (BACs), bacterial P1 constructions, or single chromosomecDNA libraries. (See, e.g., Price, C. M. (1993) Blood Rev. 7:127-134;and Trask, B. J. (1991) Trends Genet. 7:149-154.)

Fluorescent in situ hybridization (FISH) may be correlated with otherphysical chromosome mapping techniques and genetic map data. (See, e.g.,Heinz-Ulrich, et al. (1995) in Meyers, R. A. (ed.) Molecular Biology andBiotechnology, VCH Publishers New York, N.Y., pp. 965-968.) Examples ofgenetic map data can be found in various scientific journals or at theOnline Mendelian Inheritance in Man (OMIM) site. Correlation between thelocation of the gene encoding HITLP on a physical chromosomal map and aspecific disorder, or a predisposition to a specific disorder, may helpdefine the region of DNA associated with that disorder. The nucleotidesequences of the invention may be used to detect differences in genesequences among normal, carrier, and affected individuals.

In situ hybridization of chromosomal preparations and physical mappingtechniques, such as linkage analysis using established chromosomalmarkers, may be used for extending genetic maps. Often the placement ofa gene on the chromosome of another mammalian species, such as mouse,may reveal associated markers even if the number or arm of a particularhuman chromosome is not known. New sequences can be assigned tochromosomal arms by physical mapping. This provides valuable informationto investigators searching for disease genes using positional cloning orother gene discovery techniques. Once the disease or syndrome has beencrudely localized by genetic linkage to a particular genomic region,e.g., ataxia-telangiectasia to 11q22-23, any sequences mapping to thatarea may represent associated or regulatory genes for furtherinvestigation. (See, e.g., Gatti, R. A. et al. (1988) Nature336:577-580.) The nucleotide sequence of the subject invention may alsobe used to detect differences in the chromosomal location due totranslocation, inversion, etc., among normal, carrier, or affectedindividuals.

In another embodiment of the invention, HITLP, its catalytic orimmunogenic fragments, or oligopeptides thereof can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, bome on a cell surface, or locatedintracellularly. The formation of binding complexes between HITLP andthe agent being tested may be measured.

Another technique for drug screening provides for high throughputscreening of compounds having suitable binding affinity to the proteinof interest. (See, e.g., Geysen, et al. (1984) PCT applicationWO84/03564.) In this method, large numbers of different small testcompounds are synthesized on a solid substrate, such as plastic pins orsome other surface. The test compounds are reacted with HITLP, orfragments thereof, and washed. Bound HITLP is then detected by methodswell known in the art. Purified HITLP can also be coated directly ontoplates for use in the aforementioned drug screening techniques.Alternatively, non-neutralizing antibodies can be used to capture thepeptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding HITLP specificallycompete with a test compound for binding HITLP. In this manner,antibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with HITLP.

In additional embodiments, the nucleotide sequences which encode HITLPmay be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

The examples below are provided to illustrate the subject invention andare not included for the purpose of limiting the invention.

EXAMPLES

I. BRSTNOT18 cDNA Library Construction

The BRSTNOT18 cDNA library was constructed using RNA isolated fromdiseased breast tissue removed from a 57-year old Caucasian female(specimen #0618) during a unilateral simple extended mastectomy.Pathology indicated mildly proliferative breast disease. Patient historyincluded breast cancer and osteoarthritis. Family history included TypeII diabetes, gallbladder and breast cancer, and chronic lymphocyticleukemia.

The frozen tissue was homogenized and lysed in Trizol reagent (Cat.#10296-028; LIFE TECHNOLOGIES™, Gaithersburg, Md.), a monoplasticsolution of phenol and guanidine isothiocyanate, using a BrinkmannHomogenizer Polytron PT-3000 (Brinkmann Instruments, Westbury, N.Y.).After a brief incubation on ice, chloroform was added (1:5 v/v) and thelysate was centrifuged. The upper chloroform layer was removed and theRNA extracted with isopropanol, resuspended in water, and treated withDNase for 25 min at 37° C. The RNA was re-extracted once with acidphenol-chloroform pH 4.7 and precipitated using 0.3M sodium acetate and2.5 volumes ethanol. Poly(A+) RNA was isolated using the Qiagen Oligotexkit (QIAGEN, Inc., Chatsworth, Calif.).

Poly(A+) RNA was used for cDNA synthesis and library constructionaccording to the recommended protocols in the SuperScript plasmid system(Cat. #18248-013, LIFE TECHNOLOGIES™). cDNAs were fractionated on aSepharose CL4B column (Cat. #275105-01, Pharmacia Amersham Biotech,Piscataway, N.J.) and those cDNAs exceeding 400 bp were ligated intopINCY (Incyte Pharmaceuticals, Inc., Palo Alto, Calif.) and subsequentlytransformed into DH5α™ competent cells (Cat. #18258-012, LIFETECHNOLOGEES™).

II. Isolation of cDNA Clones

Plasmid DNA was released from the cells and purified using the REAL Prep96 plasmid kit (Catalog #26173, QIAGEN, Inc.). The recommended protocolwas employed except for the following changes: 1) the bacteria werecultured in 1 ml of sterile Terrific Broth (Catalog #22711, LIFETECHNOLOGIES™) with carbenicillin at 25 mg/L and glycerol at 0.4%; 2)after the cultures were incubated for 19 hours, the cells were lysedwith 0.3 ml of lysis buffer; and 3) following isopropanol precipitation,the plasmid DNA pellets were resuspended in 0.1 ml of distilled water.The DNA samples were stored at 4° C.

III. Sequencing and Analysis

The cDNAs were prepared for sequencing using either an ABI PRISMCATALYST 800 (Perkin-Elmer Applied Biosystems, Foster City, Calif.) or aMICROLAB 2200 (Hamilton Co., Reno, Nev.) sequencing preparation systemin combination with Peltier PTC-200 thermal cyclers (MJ Research, Inc.,Watertown, Mass.). The cDNAs were sequenced using the ABI PRISM 373 or377 sequencing systems and ABI protocols, base calling software, andkits (Perkin-Elmer Applied Biosystems). Alternatively, solutions anddyes from Amersham Pharmacia Biotech, Ltd. Reading frames weredetermined using standard methods (Ausubel, supra). Some of the cDNAsequences were selected for extension using the techniques disclosed inExample V.

The polynucleotide sequences derived from cDNA, extension, and shotgunsequencing were assembled and analyzed using a combination of softwareprograms which utilize algorithms well known to those skilled in theart. Table 1 summarizes the programs used, relevant references, andthreshold parameters used. The references cited in the third column ofTable 1 are incorporated by reference herein. Sequences were alsoanalyzed using MACDNASIS PRO software (Hitachi Software Engineering Co.,Ltd. San Bruno, Calif.) and the multisequence alignment program ofLASERGENE software (DNASTAR Inc, Madison Wis.).

The polynucleotide sequences were validated by removing vector, linker,and polyA tail sequences and by masking ambiguous bases, usingalgorithms and programs based on BLAST, dynamic programing, anddinucleotide nearest neighbor analysis. The sequences were then queriedagainst a selection of public databases such as GenBank primate, rodent,mammalian, vertebrate, and eukaryote databases, and BLOCKS to acquireannotation, using programs based on BLAST, FASTA, and BLIMPS. Thesequences were assembled into full length polynucleotide sequences usingprograms based on Phred, Phrap, and Consed, and were screened for openreading frames using programs based on GeneMark, BLAST, and FASTA. Thiswas followed by translation of the full length polynucleotide sequencesto derive the corresponding full length amino acid sequences. These fulllength polynucleotide and amino acid sequences were subsequentlyanalyzed by querying against databases such as the GenBank databasesdescribed above and SwissProt, BLOCKS, PRINTS, PFAM, and Prosite.

IV. Northern Analysis

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound. (See, e.g., Sambrook, supra, ch. 7; andAusubel, supra, ch. 4 and 16.)

Electronic northerns were produced using analogous computer techniques.These techniques apply BLAST to search for identical or relatedmolecules in nucleotide databases such as GenBank or LIFESEQ™ database(Incyte Pharmaceuticals). The sensitivity of the computer search wasmodified to determine the specificity of the match. The basis of thesearch is the product score, which is defined as: ##EQU1## The productscore encompasses both the degree of similarity between two sequencesand the length of the sequence match. For example, with a product scoreof 40, the match may have a possibility of a 1% to 2% error, incontrast, a product score of 70 indicates that the match will be exact.Similar molecules were identified by product scores between 15 and 40,although lower scores may identify related molecules.

Electronic northern analysis further involved the categorization of cDNAlibraries by organ/tissue and disease. The organ/tissue categoriesincluded cardiovascular, dermatologic, developmental, endocrine,gastrointestinal, hematopoietic/immune, musculoskeletal, nervous,reproductive, and urologic. The disease categories included cancer,inflammation/trauma, fetal, neurological, and pooled. For each category,the number of libraries expressing the sequence of interest was dividedby the total number of libraries across all categories. The resultsabove were reported as a percentage distribution.

V. Extension of HITLP Encoding Polynucleotides

The full length nucleic acid sequence of SEQ ID NO:2 was produced byextension of an appropriate fragment of the full length molecule, usingoligonucleotide primers designed from this fragment. One primer wassynthesized to initiate extension of an antisense polynucleotide, andthe other was synthesized to initiate extension of a sensepolynucleotide. Primers were used to facilitate the extension of theknown sequence "outward" generating amplicons containing new unknownnucleotide sequence for the region of interest. The initial primers weredesigned from the cDNA using OLIGO™ 4.06 (National Biosciences,Plymouth, Minn.), or another appropriate program, to be about 22 to 30nucleotides in length, to have a GC content of about 50% or more, and toanneal to the target sequence at temperatures of about 68° C. to about72° C. Any stretch of nucleotides which would result in hairpinstructures and primer--primer dimerizations was avoided.

Selected human cDNA libraries (GIBCO BRL) were used to extend thesequence. If more than one extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

High fidelity amplification was obtained by following the instructionsfor the XL-PCR™ kit (The Perkin-Elmer Corp., Norwalk, Conn.) andthoroughly mixing the enzyme and reaction mix. PCR was performed usingthe PTC-200 thermal cycler (MJ Research, Inc., Watertown, Mass.),beginning with 40 pmol of each primer and the recommended concentrationsof all other components of the kit, with the following parameters:

    ______________________________________                                        Step 1   94° C. for 1 min (initial denaturation)                       Step 2   65° C. for 1 min                                              Step 3   68° C. for 6 min                                              Step 4   94° C. for 15 sec                                             Step 5   65° C. for 1 min                                              Step 6   68° C. for 7 min                                              Step 7   Repeat steps 4 through 6 for an additional 15 cycles                 Step 8   94° C. for 15 sec                                             Step 9   65° C. for 1 min                                              Step 10  68° C. for 7:15 min                                           Step 11  Repeat steps 8 through 10 for an additional 12 cycles                Step 12  72° C. for 8 min                                              Step 13  4° C. (and holding)                                           ______________________________________                                    

A 5 μl to 10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a low concentration (about 0.6% to 0.8%) agarosemini-gel to determine which reactions were successful in extending thesequence. Bands thought to contain the largest products were excisedfrom the gel, purified using QIAQUICK™ (QIAGEN Inc.), and trimmed ofoverhangs using Klenow enzyme to facilitate religation and cloning.

After ethanol precipitation, the products were redissolved in 13 μl ofligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase were added, and the mixture was incubated at roomtemperature for 2 to 3 hours, or overnight at 16° C. Competent E. colicells (in 40 8 μl of appropriate media) were transformed with 3 μl ofligation mixture and cultured in 80 μl of SOC medium. (See, e.g.,Sambrook, supra, Appendix A, p. 2.) After incubation for one hour at 37°C., the E. coli mixture was plated on Luria Bertani (LB) agar (See,e.g., Sambrook, supra, Appendix A, p. 1) containing carbenicillin (2×carb). The following day, several colonies were randomly picked fromeach plate and cultured in 150 μl of liquid LB/2× carb medium placed inan individual well of an appropriate commercially-available sterile96-well microtiter plate. The following day, 5 μl of each overnightculture was transferred into a non-sterile 96-well plate and, afterdilution 1:10 with water, 5 μl from each sample was transferred into aPCR array.

For PCR amplification, 18 μl of concentrated PCR reaction mix (3.3×)containing 4 units of rTth DNA polymerase, a vector primer, and one orboth of the gene specific primers used for the extension reaction wereadded to each well. Amplification was performed using the followingconditions:

    ______________________________________                                        Step 1    94° C. for 60 sec                                            Step 2    94° C. for 20 sec                                            Step 3    55° C. for 30 sec                                            Step 4    72° C. for 90 sec                                            Step 5    Repeat steps 2 through 4 for an additional 29 cycles                Step 6    72° C. for 180 sec                                           Step 7    4° C. (and holding)                                          ______________________________________                                    

Aliquots of the PCR reactions were run on agarose gels together withmolecular weight markers. The sizes of the PCR products were compared tothe original partial cDNAs, and appropriate clones were selected,ligated into plasmid, and sequenced.

In like manner, the nucleotide sequence of SEQ ID NO:2 is used to obtain5' regulatory sequences using the procedure above, oligonucleotidesdesigned for 5' extension, and an appropriate genomic library.

VI. Labeling and Use of Individual Hybridization Probes

Hybridization probes derived from SEQ ID NO:2 are employed to screencDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base pairs, is specificallydescribed, essentially the same procedure is used with larger nucleotidefragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO™ 4.06 software (National Biosciences) and labeled bycombining 50 pmol of each oligomer, 250 μCi of [γ-³² P] adenosinetriphosphate (Amersham, Chicago, Ill.), and T4 polynucleotide kinase(DuPont NEN®, Boston, Mass.). The labeled oligonucleotides aresubstantially purified using a Sephadex™ G-25 superfine size exclusiondextran bead column (Pharmacia & Upjohn, Kalamazoo, Mich.). An aliquotcontaining 10⁷ counts per minute of the labeled probe is used in atypical membrane-based hybridization analysis of human genomic DNAdigested with one of the following endonucleases: Ase I, Bgl II, Eco RI,Pst I, Xbal, or Pvu II (DuPont NEN, Boston, Mass.).

The DNA from each digest is fractionated on a 0.7% agarose gel andtransferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham, N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1 × salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR™ film(Kodak, Rochester, N.Y.) is exposed to the blots to film for severalhours, hybridization patterns are compared visually.

VII. Microarrays

A chemical coupling procedure and an ink jet device can be used tosynthesize array elements on the surface of a substrate. (See, e.g.,Baldeschweiler, supra.) An array analogous to a dot or slot blot mayalso be used to arrange and link elements to the surface of a substrateusing thermnal, UV, chemical, or mechanical bonding procedures. Atypical array may be produced by hand or using available methods andmachines and contain any appropriate number of elements. Afterhybridization, nonhybridized probes are removed and a scanner used todetermine the levels and patterns of fluorescence. The degree ofcomplementarity and the relative abundance of each probe whichhybridizes to an element on the microarray may be assessed throughanalysis of the scanned images.

Full-length cDNAs, Expressed Sequence Tags (ESTs), or fragments thereofmay comprise the elements of the microarray. Fragments suitable forhybridization can be selected using software well known in the art suchas LASERGENE™. Full-length cDNAs, ESTs, or fragments thereofcorresponding to one of the nucleotide sequences of the presentinvention, or selected at random from a cDNA library relevant to thepresent invention, are arranged on an appropriate substrate, e.g., aglass slide. The cDNA is fixed to the slide using, e.g., UVcross-linking followed by thermal and chemical treatments and subsequentdrying. (See, e.g., Schena, M. et al. (1995) Science 270:467-470; andShalon, D. et al. (1996) Genome Res. 6:639-645.) Fluorescent probes areprepared and used for hybridization to the elements on the substrate.The substrate is analyzed by procedures described above.

VIII. Complementary Polynucleotides

Sequences complementary to the HITLP-encoding sequences, or any partsthereof, are used to detect, decrease, or inhibit expression ofnaturally occurring HITLP. Although use of oligonucleotides comprisingfrom about 15 to 30 base pairs is described, essentially the sameprocedure is used with smaller or with larger sequence fragments.Appropriate oligonucleotides are designed using OLIGO™ 4.06 software andthe coding sequence of HITLP. To inhibit transcription, a complementaryoligonucleotide is designed from the most unique 5' sequence and used toprevent promoter binding to the coding sequence. To inhibit translation,a complementary oligonucleotide is designed to prevent ribosomal bindingto the HITLP-encoding transcript.

IX. Expression of HITLP

Expression and purification of HITLP is achieved using bacterial orvirus-based expression systems. For expression of HITLP in bacteria,cDNA is subcloned into an appropriate vector containing an antibioticresistance gene and an inducible promoter that directs high levels ofcDNA transcription. Examples of such promoters include, but are notlimited to, the trp-lac (tac) hybrid promoter and the T5 or T7bacteriophage promoter in conjunction with the lac operator regulatoryelement. Recombinant vectors are transformed into suitable bacterialhosts, e.g., BL21(DE3). Antibiotic resistant bacteria express HITLP uponinduction with isopropyl beta-D-thiogalactopyranoside (IPTG). Expressionof HITLP in eukaryotic cells is achieved by infecting insect ormammalian cell lines with recombinant Autographica californica nuclearpolyhedrosis virus (AcMNPV), commonly known as baculovirus. Thenonessential polyhedrin gene of baculovirus is replaced with cDNAencoding HITLP by either homologous recombination or bacterial-mediatedtransposition involving transfer plasmid intermediates. Viralinfectivity is maintained and the strong polyhedrin promoter drives highlevels of cDNA transcription. Recombinant baculovirus is used to infectSpodoptera frugiperda (Sf9) insect cells in most cases, or humanhepatocytes, in some cases. Infection of the latter requires additionalgenetic modifications to baculovirus. (See Engelhard, E. K. et al.(1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996)Hum. Gene Ther. 7:1937-1945.)

In most expression systems, HITLP is synthesized as a fusion proteinwith, e.g., glutathione S-transferase (GST) or a peptide epitope tag,such as FLAG or 6-His, permitting rapid, single-step, affinity-basedpurification of recombinant fusion protein from crude cell lysates. GST,a 26-kilodalton enzyme from Schistosoma japonicum, enables thepurification of fusion proteins on immobilized glutathione underconditions that maintain protein activity and antigenicity (Pharmacia,Piscataway, N.J.). Following purification, the GST moiety can beproteolytically cleaved from HITLP at specifically engineered sites.FLAG, an 8-amino acid peptide, enables immunoaffinity purification usingcommercially available monoclonal and polyclonal anti-FLAG antibodies(Eastman Kodak, Rochester, N.Y.). 6-His, a stretch of six consecutivehistidine residues, enables purification on metal-chelate resins (QIAGENInc, Chatsworth, Calif.). Methods for protein expression andpurification are discussed in Ausubel, F. M. et al. (1995 and periodicsupplements) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y., ch 10, 16. Purified HITLP obtained by these methods canbe used directly in the following activity assay.

X. Demonstration of HITLP Activity

HITLP activity is measured as the ability to produce a short-circuitcurrent by stimulating ion transport across a target epithelium.(Meredith, supra.) Epithelium from target tissue is mounted as a flatsheet between Ussing chambers containing 2 ml of physiological saline,resembling extracellular fluid in its composition, at 37 ° C. The salineis oxygenated and mixed by bubbling with 95% oxygen, 5% carbon dioxide.The short-circuit current (I_(SC) ; μA.cm²), measured using a voltageclamp system, is allowed to fall close to 0 mV (baseline). HITLP orcontrol solution is then added to the basal side of the epithelial sheetand the open-circuit transepithelial potential is measured at intervals.The increase in I_(SC) caused by HITLP is a direct measure of activetransport of Cl⁻ from the lumenal side. HITLP activity is measured inμequivalents per hour-cm².

XI. Functional Assays

HITLP function is assessed by expressing the sequences encoding HITLP atphysiologically elevated levels in mammalian cell culture systems. cDNAis subcloned into a mammalian expression vector containing a strongpromoter that drives high levels of cDNA expression. Vectors of choiceinclude pCMV SPORT™ (Life Technologies, Gaithersburg, Md.) and pCR™ 3.1(Invitrogen, Carlsbad, Calif., both of which contain the cytomegaloviruspromoter. 5-10 μg of recombinant vector are transiently transfected intoa human cell line, preferably of endothelial or hematopoietic origin,using either liposome formulations or electroporation. 1-2 μg of anadditional plasmid containing sequences encoding a marker protein areco-transfected. Expression of a marker protein provides a means todistinguish transfected cells from nontransfected cells and is areliable predictor of cDNA expression from the recombinant vector.Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP)(Clontech, Palo Alto, Calif.), CD64, or a CD64-GFP fusion protein. Flowcytometry (FCM), an automated, laser optics-based technique, is used toidentify transfected cells expressing GFP or CD64-GFP, and to evaluateproperties, for example, their apoptotic state. FCM detects andquantifies the uptake of fluorescent molecules that diagnose eventspreceding or coincident with cell death. These events include changes innuclear DNA content as measured by staining of DNA with propidiumiodide; changes in cell size and granularity as measured by forwardlight scatter and 90 degree side light scatter; down-regulation of DNAsynthesis as measured by decrease in bromodeoxyuridine uptake;alterations in expression of cell surface and intracellular proteins asmeasured by reactivity with specific antibodies; and alterations inplasma membrane composition as measured by the binding offluorescein-conjugated Annexin V protein to the cell surface. Methods inflow cytometry are discussed in Ormerod, M. G. (1 994) Flow Cytometry,Oxford, New York, N.Y.

The influence of HITLP on gene expression can be assessed using highlypurified populations of cells transfected with sequences encoding HITLPand either CD64 or CD64-GFP. CD64 and CD64-GFP are expressed on thesurface of transfected cells and bind to conserved regions of humanimmunoglobulin G (IgG). Transfected cells are efficiently separated fromnontransfected cells using magnetic beads coated with either human IgGor antibody against CD64 (DYNAL, Lake Success, N.Y.). mRNA can bepurified from the cells using methods well known by those of skill inthe art. Expression of mRNA encoding HITLP and other genes of interestcan be analyzed by Northern analysis or microarray techniques.

XII. Production of HITLP Specific Antibodies

HITLP substantially purified using polyacrylamide gel electrophoresis(PAGE)(see, e.g., Harrington, M. G. (1990) Methods Enzymol.182:488-495), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols.

Alternatively, the HITLP amino acid sequence is analyzed usingLASERGENE™ software (DNASTAR Inc.) to determine regions of highimmunogenicity, and a corresponding oligopeptide is synthesized and usedto raise antibodies by means known to those of skill in the art. Methodsfor selection of appropriate epitopes, such as those near the C-terminusor in hydrophilic regions are well described in the art. (See, e.g.,Ausubel supra, ch. 11.)

Typically, oligopeptides 15 residues in length are synthesized using anApplied Biosystems Peptide Synthesizer Model 431 A using fmoc-chemistryand coupled to KLH (Sigma, St. Louis, Mo.) by reaction withN-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increaseimmunogenicity. (See, e.g., Ausubel supra.) Rabbits are immunized withthe oligopeptide-KLH complex in complete Freund's adjuvant. Resultingantisera are tested for antipeptide activity by, for example, bindingthe peptide to plastic, blocking with 1% BSA, reacting with rabbitantisera, washing, and reacting with radio-iodinated goat anti-rabbitIgG.

XIII. Purification of Naturally Occurring HITLP Using SpecificAntibodies

Naturally occurring or recombinant HITLP is substantially purified byimmunoaffinity chromatography using antibodies specific for HITLP. Animmunoaffinity column is constructed by covalently coupling anti-HITLPantibody to an activated chromatographic resin, such as CNBr-activatedSepharose (Pharmacia & Upjohn). After the coupling, the resin is blockedand washed according to the manufacturer's instructions.

Media containing HITLP are passed over the immunoaffinity column, andthe column is washed under conditions that allow the preferentialabsorbance of HITLP (e.g., high ionic strength buffers in the presenceof detergent). The column is eluted under conditions that disruptantibody/HITLP binding (e.g., a buffer of pH 2 to pH 3, or a highconcentration of a chaotrope, such as urea or thiocyanate ion), andHITLP is collected.

XIV. Identification of Molecules Which Interact with HITLP

HITLP, or biologically active fragments thereof, are labeled with ¹²⁵ IBolton-Hunter reagent. (See, e.g., Bolton et al. (1973) Biochem. J.133:529.) Candidate molecules previously arrayed in the wells of amulti-well plate are incubated with the labeled HITLP, washed, and anywells with labeled HITLP complex are assayed. Data obtained usingdifferent concentrations of HITLP are used to calculate values for thenumber, affinity, and association of HITLP with the candidate molecules.

Various modifications and variations of the described methods andsystems of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the followingclaims.

                                      TABLE 1                                     __________________________________________________________________________    Program Desription          Reference           Parameter                     __________________________________________________________________________                                                    Threshold                     ABI FACTURA                                                                           A program that removes vector sequences and                                                       Perkin-Elmer Applied Biosystems,                          masks ambiguous bases in nucleic acid                                                             Foster City, CA.                                          sequences.                                                            ABI/PARACEL                                                                           A Fast Data Finder useful in comparing and                                                        Perkin-Elmer Applied Biosystems,                                                                  Mismatch <50%                 FDF     annotating amino acid or nucleic acid                                                             Foster City, CA; Paracel Inc., Pasadena, CA.              sequences.                                                            ABI     A program that assembles nucleic acid                                                             Perkin-Elmer Applied Biosystems,                  AutoAssembler                                                                         sequences.          Foster City, CA.                                  BLAST   A Basic Local Alignment Search Tool useful in                                                     Altschul, S. F. et al. (1990) J. Mol.                                                             ESTs: Probability value =                                                     1.0E-8                                sequence similarity search for amino acid and                                                     215:403-410; Altschul, S. F. et al.                                                               or less                               nucleic acid sequences. BLAST includes five                                                       Nucleic Acids Res. 25: 3389-3402.                                                                 Full Length sequences:                                                        Probability                           functions: blastp, blastn, blastx, tblastn,                                                                           value = 1.0E-10 or less               and tblastx.                                                          FASTA   A Pearson and Lipman algorithm that searches                                                      Pearson, W. R. and D. J. Lipman (1988)                                                            ESTs: fasta E value =                                                         1.06E-6                               for similarity between a query sequence and a                                                     Natl. Acad Sci. 85:2444-2448; Pearson, W.                                                         Assembled ESTs: fasta                                                         Identity =                            group of sequences of the same type. FASTA                                                        (1990) Methods Enzymol. 183: 63-98;                                                               95% or greater and Match              comprises as least five functions: fasta,                                                         Smith, T. F. and M. S. Waterman (1981)                                                            length = 200 bases or                                                         greater; fastx                        tfasta, fastx, tfastx, and ssearch.                                                               Appl. Math. 2:482-489.                                                                            E value = 1.0E-8 or less                                                      Full Length sequences:                                                        fastx                                                                         score = 100 or greater        BLIMPS  A BLocks IMProved Searcher that matches a                                                         Henikoff, S and J. G. Henikoff, Nucl.                                                             Score = 1000 or greater;                                                      Ratio of                              sequence against those in BLOCKS and                                                              Res., 19:6565-72, 1991. J. G. Henikoff and                                                        Score/Strength = 0.75 or                                                      larger;                               PRINTS databases to search for gene families,                                                     Henikoff (1996) Methods Enzymol.                                                                  and Probability value =                                                       1.0E-3 or                             sequence homology, and structural fingerprint                                                     105; and Attwood, T. K. et al. (1997) J.                                      Chem.               less                                  regions.            Inf. Comput. Sci. 37: 417-424.                    PFAM    A Hidden Markov Models-based application                                                          Krogh, A. et al. (1994) J. Mol.                                                                   Score = 10-50 bits,                                                           depending on                          useful for protein family search.                                                                 235:1501-1531; Sonnhammer, E. L. L. et                                                            individual protein                                                            families                                                  (1988) Nucleic Acids Res. 26:320-322.             ProfileScan                                                                           An algorithm that searches for structural and                                                     Gribskov, M. et al. (1988) CABIOS                                                                 Score = 4.0 or greater                sequence motifs in protein sequences that                                                         Gribskov, et al. (1989) Methods Enzymol.                  match sequence patterns defined in Prosite.                                                       183:146-159; Bairoch, A. et al. (1997)                                        Nucleic                                                                       Acids Res. 25: 217-221.                           Phred   A base-calling algorithm that examines auto-                                                      Ewing, B. et al. (1998) Genome                            mated sequencer traces with high sensitivity                                                      Res. 8:175-185; Ewing, B. and P.                          and probability.    Green (1998) Genome Res. 8:186-194.               Phrap   A Phils Revised Assembly Program including                                                        Smith, T. F. and M. S. Waterman (1981)                                                            Score = 120 or greater;                                                       Match                                 SWAT and CrossMatch, programs based on                                                            Appl. Math. 2:482-489; Smith, T. F. and M.                                                        length = 56 or greater                efficient implementation of the                                                                   Waterman (1981) J. Mol. Biol. 147:195-197;                Smith-Waterman algorithm, useful in searching                                                     and Green, P., University of Washington,                  sequence homology and assembling DNA                                                              Seattle, WA.                                              sequences.                                                            Consed  A graphical tool for viewing and editing Phrap                                                    Gordon, D. et al. (1998) Genome                           assemblies          Res. 8:195-202.                                   SPScan  A weight matrix analysis program that scans                                                       Nielson, H. et al. (1997) Protein                                                                 Score = 5 or greater                  protein sequences for the presence of secretory                                                   10:1-6; Claverie, J. M. and S. Audic (1997)               signal peptides.    CABIOS 12: 431-439.                               Motifs  A program that searches amino acid sequences                                                      Bairoch et al. supra; Wisconsin                           for patterns that matched those defined in                                                        Package Program Manual, version                           Prosite.            9, page M51-59, Genetics Computer                                             Group, Madison, WI.                               __________________________________________________________________________

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - <160> NUMBER OF SEQ ID NOS: 4                                               - <210> SEQ ID NO 1                                                           <211> LENGTH: 109                                                             <212> TYPE: PRT                                                               <213> ORGANISM: HOMO SAPIENS                                                  <220> FEATURE:                                                                <223> OTHER INFORMATION: 3171334, BRSTNOT18                                   - <400> SEQUENCE: 1                                                           - Met Met Cys Ser Arg Asn Ile Lys Ile Ser Va - #l Val Leu Ser Leu Val         #                15                                                           - Leu Ile Pro Ile Phe Ala Ala Leu Pro His As - #n His Asn Leu Ser Lys         #            30                                                               - Arg Ser Asn Phe Phe Asp Leu Glu Cys Lys Gl - #y Ile Phe Asn Lys Thr         #        45                                                                   - Met Phe Phe Arg Leu Asp Arg Ile Cys Glu As - #p Cys Tyr Gln Leu Phe         #    60                                                                       - Arg Glu Thr Ser Ile His Arg Leu Cys Lys Gl - #n Glu Cys Phe Gly Ser         #80                                                                           - Pro Phe Phe Asn Ala Cys Ile Glu Ala Leu Gl - #n Leu His Glu Glu Met         #                95                                                           - Asp Lys Tyr Asn Glu Trp Arg Asp Thr Leu Gl - #y Arg Lys                     #           105                                                               - <210> SEQ ID NO 2                                                           <211> LENGTH: 554                                                             <212> TYPE: DNA                                                               <213> ORGANISM: HOMO SAPIENS                                                  <220> FEATURE:                                                                <223> OTHER INFORMATION: 3171334, BRSTNOT18                                   - <400> SEQUENCE: 2                                                           - tagactcgtc gggaaagtgc tcactaagtt cattccgctc atccgtcaag cc - #agcgaaag         60                                                                          - gtcctcaata tgatgtgttc ccgcaacata aagatctcgg tggtgctgtc tc - #ttgtcctg        120                                                                          - ataccaatct tcgccgcctt gccacacaac cacaatctgt cgaagcgcag ca - #acttcttc        180                                                                          - gacctggagt gcaagggcat cttcaacaag accatgttct tccgactgga cc - #gcatctgc        240                                                                          - gaggactgct accagttgtt ccgcgagacg agtatacacc gattatgcaa gc - #aagaatgc        300                                                                          - ttcggatcgc ccttcttcaa cgcctgcata gaggctcttc agctgcacga gg - #agatggac        360                                                                          - aagtataacg aatggcgcga taccctgggt cgcaagtaaa gtgcgattct ct - #gggatttt        420                                                                          - ctggcaccgt cactggcacg agcagctact taattctatg aactattaac ta - #aaactatt        480                                                                          - attattgata actgtggcag aacacggcac gaaactatgg caaccaggaa ac - #gatcgacc        540                                                                          #    554                                                                      - <210> SEQ ID NO 3                                                           <211> LENGTH: 130                                                             <212> TYPE: PRT                                                               <213> ORGANISM: SCHISTOCERCA GREGARIA                                         <220> FEATURE:                                                                <223> OTHER INFORMATION: 1244522, GENBANK                                     - <400> SEQUENCE: 3                                                           - Met His His Gln Lys Gln Gln Gln Gln Gln Ly - #s Gln Gln Gly Glu Ala         #                15                                                           - Pro Cys Arg His Leu Gln Trp Arg Leu Ser Gl - #y Val Val Leu Cys Val         #            30                                                               - Leu Val Val Ala Ser Leu Val Ser Thr Ala Al - #a Ser Ser Pro Leu Asp         #        45                                                                   - Pro His His Leu Ala Lys Arg Ser Phe Phe As - #p Ile Gln Cys Lys Gly         #    60                                                                       - Val Tyr Asp Lys Ser Ile Phe Ala Arg Leu As - #p Arg Ile Cys Glu Asp         #80                                                                           - Cys Tyr Asn Leu Phe Arg Glu Pro Gln Leu Hi - #s Ser Leu Cys Arg Ser         #                95                                                           - Asp Cys Phe Lys Ser Pro Tyr Phe Lys Gly Cy - #s Leu Gln Ala Leu Leu         #           110                                                               - Leu Ile Asp Glu Glu Glu Lys Phe Asn Gln Me - #t Val Glu Ile Leu Gly         #       125                                                                   - Lys Lys                                                                         130                                                                       - <210> SEQ ID NO 4                                                           <211> LENGTH: 134                                                             <212> TYPE: PRT                                                               <213> ORGANISM: SCHISTOCERCA GREGARIA                                         <220> FEATURE:                                                                <223> OTHER INFORMATION: 1244524, GENBANK                                     - <400> SEQUENCE: 4                                                           - Met His His Gln Lys Gln Gln Gln Gln Gln Ly - #s Gln Gln Gly Glu Ala         #                15                                                           - Pro Cys Arg His Leu Gln Trp Arg Leu Ser Gl - #y Val Val Leu Cys Val         #            30                                                               - Leu Val Val Ala Ser Leu Val Ser Thr Ala Al - #a Ser Ser Pro Leu Asp         #        45                                                                   - Pro His His Leu Ala Lys Arg Ser Phe Phe As - #p Ile Gln Cys Lys Gly         #    60                                                                       - Val Tyr Asp Lys Ser Ile Phe Ala Arg Leu As - #p Arg Ile Cys Glu Asp         #80                                                                           - Cys Tyr Asn Leu Phe Arg Glu Pro Gln Leu Hi - #s Ser Leu Cys Arg Lys         #                95                                                           - Asp Cys Phe Thr Ser Asp Tyr Phe Lys Gly Cy - #s Ile Asp Val Leu Leu         #           110                                                               - Leu Gln Asp Asp Met Asp Lys Ile Gln Ser Tr - #p Ile Lys Gln Ile His         #       125                                                                   - Gly Ala Glu Pro Gly Val                                                         130                                                                       __________________________________________________________________________

What is claimed is:
 1. An isolated and purified polynucleotide fragmentencoding the polypeptide of SEQ ID NO:1 or residues 99-105 of SEQ IDNO:1.
 2. An isolated and purified polynucleotide which is completelycomplementary to the polynucleotide of SEQ ID NO:1.
 3. An isolated andpurified polynucleotide fragment comprising the polynucleotide sequenceof SEQ ID NO:2 or nucleotides 364-384 of SEQ ID NO:2.
 4. An isolated andpurified polynucleotide which is completely complementary to thepolynucleotide of SEQ ID NO:2.
 5. An expression vector comprising thepolynucleotide fragment of claim
 1. 6. A host cell comprising theexpression vector of claim
 5. 7. A method for producing a polypeptidecomprising the sequence of SEQ ID NO:1 or residues 99-105 of SEQ IDNO:1, the method comprising the steps of:a) culturing the host cell ofclaim 6 under conditions suitable for the expression of thepolynucleotide; and b) recovering the polypeptide from the host cellculture.